U.S. patent application number 11/066136 was filed with the patent office on 2007-05-17 for protein-acrylamide copolymer hydrogels and method of using same to measure protein concentration and activity.
Invention is credited to Shawn B. Brueggemeier, Stephen J. Kron, Sean P. Palecek.
Application Number | 20070111273 11/066136 |
Document ID | / |
Family ID | 38041360 |
Filed Date | 2007-05-17 |
United States Patent
Application |
20070111273 |
Kind Code |
A1 |
Brueggemeier; Shawn B. ; et
al. |
May 17, 2007 |
Protein-acrylamide copolymer hydrogels and method of using same to
measure protein concentration and activity
Abstract
Disclosed are a polyacrylamide-based method of fabricating
surface-bound peptide and protein arrays, the arrays themselves,
and a method of using the arrays to detect proteins and to measure
their concentration, binding affinity, and kinetics. Peptides,
proteins, fusion proteins, protein complexes, and the like, are
labeled with an acrylic moiety and attached to
acrylic-functionalized glass surfaces through a copolymerization
with acrylic monomer. The specific attachment of GST-green
fluorescent protein (GFP) fusion protein was more than 7-fold
greater than the nonspecific attachment of non-acrylic labeled
GST-GFP. Surface-attached GST-GFP (0.32 ng /mm.sup.2) was
detectable by direct measurement of GFP fluorescence and this lower
detection limit was reduced to 0.080 ng /mm.sup.2 using indirect
antibody-based detection. The polyacrylamide-based surface
attachment strategy was also used to measure the kinetics of
substrate phosphorylation by the kinase c-Src. The surface
attachment strategy is applicable to the proteomics field and
addresses denaturation and dehydration problems associated with
protein microarray development.
Inventors: |
Brueggemeier; Shawn B.;
(Madison, WI) ; Kron; Stephen J.; (Oak Park,
IL) ; Palecek; Sean P.; (Madison, WI) |
Correspondence
Address: |
DEWITT ROSS & STEVENS S.C.
8000 EXCELSIOR DR
SUITE 401
MADISON
WI
53717-1914
US
|
Family ID: |
38041360 |
Appl. No.: |
11/066136 |
Filed: |
February 24, 2005 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
60547198 |
Feb 24, 2004 |
|
|
|
Current U.S.
Class: |
435/23 ;
427/2.11; 435/287.1 |
Current CPC
Class: |
C07K 17/14 20130101;
G01N 33/54353 20130101 |
Class at
Publication: |
435/023 ;
435/287.1; 427/002.11 |
International
Class: |
C12Q 1/37 20060101
C12Q001/37; C12M 3/00 20060101 C12M003/00 |
Goverment Interests
FEDERAL FUNDING STATEMENT
[0002] The invention was made with United States government support
awarded by the following agencies: NSF 0103348. The United States
has certain rights in this invention.
Claims
1. A method of attaching peptides, proteins, or protein-containing
complexes to a surface, the method comprising: (a) providing a
surface having immobilized thereon at least one domain comprising a
first polymerizable ethylene-containing moiety; (b) providing a
functionalized peptide, protein, or protein-containing complex
modified to include a second polymerizable ethylene-containing
moiety; and then (c) polymerizing the functionalized peptide,
protein, or protein-containing complex of step (b) with the domain
of step (a), such that the peptide, protein, or protein-containing
complex is immobilized upon or within the domain.
2. The method according to claim 1, wherein step (a) comprises
providing a surface having immobilized thereon at least one domain
comprising a gel selected from the group consisting of acrylic
acid-, alkylacrylic acid-, acrylamide-, and alkylacrylamide-based
gels.
3. The method according to claim 1, wherein step (b) comprises
providing an acrylic-, alkylacrylic-, acrylamide-, or
alkylacrylamide-functionalized peptide, protein, or
protein-containing complex.
4. The method according to claim 1, wherein step (a) comprises
providing a surface having immobilized thereon at least one domain
comprising a gel selected from the group consisting of acrylic
acid-, alkylacrylic acid-, acrylamide-, and alkylacrylamide-based
gels; and step (b) comprises providing an acrylic-, alkylacrylic-,
acrylamide-, or alkylacrylamide-functionalized peptide, protein, or
protein-containing complex.
5. The method of claim 4, wherein step (b) comprises providing a
peptide, protein, or protein-containing complex which is an enzyme
or a fragment thereof, an enzyme substrate or a fragment thereof,
or a fusion protein or a fragment thereof.
6. The method of claim 1, wherein step (b) comprises providing a
peptide, protein, or protein-containing complex which is an enzyme
or a fragment thereof, an enzyme substrate or a fragment thereof,
or a fusion protein or a fragment thereof.
7. The method of claim 1, wherein step (c) comprises immobilizing
the functionalized peptide, protein, or protein-containing upon or
within the domain such that the functionalized peptide, protein, or
protein-containing complex remains accessible to participate in
chemical or enzymatic reactions, thereby yielding a reactive
surface.
8. The method of claim 7, further comprising, after step (c): (d)
contacting the reactive surface of step (c) with a reagent mixture
to be assayed and detecting whether the functionalized peptide,
protein, or protein-containing complex within the reactive surface
reacts with the reagent mixture.
9. The method of claim 1, wherein step (c) comprises polymerizing
the functionalized peptide, protein, or protein-containing complex
of step (b) with the domain of step (a) via UV-induced
polymerization or chemical-induced free-radical polymerization.
10. The method of claim 1, wherein step (a) comprises providing a
glass surface.
11. A method of attaching a peptide, protein, and/or
protein-containing complex to a surface, the method comprising: (a)
providing a surface having immobilized thereon at least one domain
comprising an acrylic acid-, alkylacrylic acid-, acrylamide-, or
alkylacrylamide-based gel; (b) providing an acrylic-,
alkylacrylic-, acrylamide-, or alkylacrylamide-functionalized
peptide, protein, or protein-containing complex; and then (c)
co-polymerizing the functionalized peptide, protein, or
protein-containing complex of step (b) with the domain of step (a),
such that the peptide, protein, or protein-containing complex is
immobilized upon or within the domain.
12. The method of claim 11, wherein step (c), comprises
immobilizing the functionalized peptide, protein, or
protein-containing complex upon or within the domain such that the
functionalized peptide, protein, or protein-containing complex
remains accessible to participate in chemical or enzymatic
reactions, thereby yielding a reactive surface.
13. The method of claim 12, further comprising, after step (c): (d)
contacting the reactive surface of step (c) with a reagent mixture
to be assayed and detecting whether the functionalized peptide,
protein, or protein-containing complex within the reactive surface
reacts with the reagent mixture.
14. The method of claim 11, wherein step (b) comprises providing a
peptide, protein, or protein-containing complex which is an enzyme
or a fragment thereof, an enzyme substrate or a fragment thereof,
or a fusion protein or a fragment thereof.
15. The method of claim 11, wherein step (c) comprises polymerizing
the functionalized peptide, protein, or protein-containing complex
of step (b) with the domain of step (a) via UV-induced
polymerization or chemical-induced free-radical polymerization.
16. A method of attaching peptides, proteins, or protein-containing
complexes to a surface and assaying the presence or activity of the
peptide, protein, and/or protein-containing complex, the method
comprising: (a) providing a surface having immobilized thereon at
least one domain comprising a first polymerizable
ethylene-containing moiety; (b) providing a peptide, protein, or
protein-containing complex modified to include a second
polymerizable ethylene-containing moiety; and then (c) polymerizing
the functionalized peptide, protein, or protein-containing complex
of step (b) with the domain of step (a) such that the peptide,
protein, or protein-containing complex is: (i) immobilized upon or
within the domain, and (ii) remains accessible to participate in
chemical or enzymatic reactions, thereby yielding a reactive
surface; and then (d) contacting the reactive surface of step (c)
with a reagent mixture to be assayed and detecting whether the
functionalized peptide, protein, or protein-containing complex
within the reactive surface reacts with the reagent mixture.
17. The method according to claim 16, wherein step (a) comprises
providing a surface having immobilized thereon at least one domain
comprising a gel selected from the group consisting of acrylic
acid-, alkylacrylic acid-, acrylamide-, and alkylacrylamide-based
gels.
18. The method according to claim 16, wherein step (b) comprises
providing an acrylic-, alkylacrylic-, acrylamide-, or
alkylacrylamide-functionalized peptide, protein, or
protein-containing complex.
19. The method according to claim 16, wherein step (a) comprises
providing a surface having immobilized thereon at least one domain
comprising a gel selected from the group consisting of acrylic
acid-, alkylacrylic acid-, acrylamide-, and alkylacrylamide-based
gels; and step (b) comprises providing an acrylic-, alkylacrylic-,
acrylamide-, or alkylacrylamide-functionalized peptide, protein, or
protein-containing complex.
20. The method of claim 19, wherein step (b) comprises providing a
peptide, protein, or protein-containing complex which is an enzyme
or a fragment thereof, an enzyme substrate or a fragment thereof,
or a fusion protein or a fragment thereof.
21. The method of claim 16, wherein step (b) comprises providing a
peptide, protein, or protein-containing complex which is an enzyme
or a fragment thereof, an enzyme substrate or a fragment thereof,
or a fusion protein or a fragment thereof.
22. The method of claim 16, wherein step (d) comprises detecting
the reaction via a method selected from the group consisting of
fluorescent, chemiluminesent, calorimetric, antibody, and
radioactive labeling, mass spectrometry and flow cytometry.
23. A composition of matter produced by: (a) providing a surface
having immobilized thereon at least one domain comprising a first
polymerizable ethylene-containing moiety; (b) providing a peptide,
protein, or protein-containing complex modified to include a second
polymerizable ethylene-containing moiety; and then (c) polymerizing
the functionalized peptide, protein, or protein-containing complex
of step (b) with the domain of step (a), such that the peptide,
protein, or protein-containing complex is immobilized upon or
within the domain.
24. A composition of matter produced by: (a) providing a surface
having immobilized thereon at least one domain comprising an
acrylic acid-, alkylacrylic acid-, acrylamide-, or
alkylacrylamide-based gel; (b) providing an acrylic-,
alkylacrylic-, acrylamide-, or alkylacrylamide-functionalized
peptide, protein, or protein-containing complex; and then (c)
co-polymerizing the functionalized peptide, protein, or
protein-containing complex of step (b) with the domain of step (a),
such that the peptide, protein, or protein-containing complex is
immobilized upon or within the domain.
25. A method of detecting presence or activity of an enzyme, the
method comprising: the method comprising: (a) providing a
composition of matter as recited claim 23 or 24, wherein the
peptide, protein, or protein-containing complex is selected from
the group consisting of an enzyme, a substrate for an enzyme, and
combinations thereof, then (b) contacting the composition of matter
of step (a) with a solution comprising or suspected of comprising a
chemical entity selected from the group consisting of a substrate
for the enzyme of step (a), an enzyme capable of catalyzing a
reaction involving the substrate of step (a), and combinations
thereof, for a time and under conditions suitable for an
enzymatically catalyzed reaction to take place; and then (c)
determining if an enzymatically catalyzed reaction took place
between the solution and the composition of matter, wherein the
presence or activity of the enzyme is measured.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] Priority is hereby claimed to provisional application Ser.
No. 60/547,198, filed Feb. 24, 2004, the entire content of which is
incorporated herein.
INTRODUCTION
[0003] Data from DNA microarrays has been used to elucidate the
control mechanisms of cells and organisms based on gene expression
profiles [1-3]. However, most cellular processes are the direct
result of protein-protein interactions. Thus, a thorough
understanding of the regulation of cellular processes must include
a parallel analysis of protein activity to supplement the gene
expression analysis. Protein activity depends on mRNA stability and
translation rates, protein stability and degradation rates,
post-translational modifications, and intracellular protein
localization. These properties do not necessarily correlate to mRNA
expression levels [4]. In short, protein analyses that rely solely
on expression profiles do not provide a complete picture of
cellular control mechanisms.
[0004] Conventionally, the majority of research efforts into
protein analysis have focused on isolating, purifying, and
characterizing a single protein or a small group of related
proteins. Recently, many groups have expanded this analysis to
include hundreds or thousands of proteins in a single experiment
through the development of protein microarrays. Microarrays of
proteins attached to nitrocellulose-coated glass slides [5],
alkanethiol-coated gold surfaces [6], poly-L-lysine-treated glass
slides [7], aldehyde-treated glass slides [8, 9], silane-modified
glass slides [10, 11], and nickel- treated glass slides [12] (among
others) have been reported. These microarray strategies cover a
wide range of specific and nonspecific protein-surface
interactions, including electrostatic attachment, affinity-based
binding, and covalent bond formation.
[0005] While numerous methods for creating protein microarrays have
been reported, a specific surface attachment strategy has yet to be
widely accepted or adopted because each of the conventional
approaches suffers from one drawback or another. For example,
adsorption-based attachment strategies yield microarrays that often
suffer specificity and homogeneity problems [13]. Other strategies
to fabricate protein microarrays require biotin- or
polyhistidine-labeled proteins. These transformations are easily
performed on a proof-of-concept scale, but are quite difficult to
scale-up to yield large microarrays suitable to analyze an entire
proteome. In addition, the close proximity of the attached proteins
to the microarray surface can sterically block potential active
sites or result in protein denaturation [9]. Denaturation due to
spot dehydration is also a concern.
[0006] In response to several of these problems, microarrays in
which oligonucleotides and proteins have been immobilized in
polyacrylamide gel pads have been investigated. Until recently,
this polyacrylamide work has focused on oligonucleotide and protein
microarrays formed by immobilizing these molecules into an
activated polyacrylamide gel matrix [14-16]. A copolymerization
procedure for immobilizing proteins has also been reported [17].
All of these prior art approaches, however, suffer from various
shortcomings.
SUMMARY OF THE INVENTION
[0007] Disclosed herein is a novel attachment method in which
peptides, proteins, fusion proteins, protein complexes, and the
like, are immobilized to modified surfaces through a
polyacrylamide-based polymerization reaction. As described in the
Examples, the characteristics of this attachment method have been
studied by examining: 1) specific and nonspecific protein
attachment; 2) stability of the protein attachment; 3) diffusion
within the polyacrylamide hydrogel spot; and 4) fluorescence and
chemiluminescence detection ranges and linearity of the resulting
signal. The kinetics of a kinase-mediated phosphorylation reaction
involving surface-attached substrates is described, along with a
study of the influence of polyacrylamide gel percentage on this
kinase reaction. The polyacrylamide-based attachment strategy
disclosed herein yields reliable and reproducible quantitative
results. The method is applicable and highly useful in a variety of
areas in the proteomic field.
[0008] Thus, one embodiment of the invention is directed to a
method of attaching a peptide, protein, and/or protein-containing
complex to a surface. The method comprises providing a surface
having immobilized thereon at least one domain comprising a first
polymerizable ethylene-containing moiety. Also provided is a
peptide, protein, or protein-containing complex modified to include
a second polymerizable ethylene-containing moiety. Then, the
functionalized peptide, protein, or protein-containing complex is
polymerized with the domain, such that the peptide, protein, or
protein-containing complex is immobilized upon and/or within the
domain.
[0009] Another embodiment of the invention is directed to a method
of assaying the presence and/or activity of a peptide, protein,
and/or protein-containing complex using the composition of matter
resulting from the method recited in the immediately preceding
paragraph. Here, the method comprises providing a surface having
immobilized thereon at least one domain comprising a first
polymerizable ethylene-containing moiety; and providing a peptide,
protein, or protein-containing complex modified to include a second
polymerizable ethylene-containing moiety. The functionalized
peptide, protein, or protein-containing complex is then polymerized
with the domain, such that the peptide, protein, or
protein-containing complex is: (i) immobilized upon and/or within
the domain, and (ii) remains accessible to participate in chemical
and enzymatic reactions; thereby yielding a reactive surface. Then,
the reactive surface is contacted with a reagent mixture to be
assayed under pre-defined reaction conditions. The reactive surface
is then examined to detect whether the functionalized peptide,
protein, or protein-containing complex within the reactive surface
reacted with the reagent mixture.
[0010] In yet another embodiment, the invention is directed to a
method of attaching a peptide, protein, and/or protein-containing
complex to a surface. The method comprises providing a surface
having immobilized thereon at least one domain comprising an
acrylic acid- or acrylamide-based gel. Also provided is an acrylic-
or acrylamide-functionalized peptide, protein, or
protein-containing complex. The functionalized peptide, protein, or
protein-containing complex is then co-polymerized with the domain
such that the peptide, protein, or protein-containing complex is
immobilized upon and/or within the domain.
[0011] In the preferred embodiment, the functionalized peptide,
protein, or protein-containing complex is immobilized upon and/or
within the domain such that it remains accessible to participate in
chemical and enzymatic reactions.
[0012] The peptide, protein, or protein-containing complex can be
any such entity, without limitation, including single-subunit and
multi-subunit proteins, metal-containing proteins, proteins
containing metallic or non-metallic prosthetic groups, etc. In a
particularly preferred embodiment, the peptide, protein, or protein
complex is an enzyme or fragment thereof, an enzyme substrate or
fragment thereof; or a fusion protein or fragment thereof.
[0013] Another embodiment of the invention is directed to a method
of assaying the presence and/or activity of a peptide, protein,
and/or protein-containing complex. Here, the method proceeds as
noted above: a surface is provided having immobilized thereon at
least one domain comprising an acrylic acid- or acrylamide-based
gel. Also provided is an acrylic- or acrylamide-functionalized
peptide, protein, or protein-containing complex. The functionalized
peptide, protein, or protein-containing complex is then
co-polymerized with the domain such that the peptide, protein, or
protein-containing complex is immobilized upon and/or within the
domain, and remains accessible to participate in chemical and
enzymatic reactions. This composition of matter is referred to
herein as the "reactive surface." The reactive surface is then
contacted with a reagent mixture to be assayed. Any reaction
between the functionalized peptide, protein, or protein-containing
complex within the reactive surface and the reagent mixture is then
detected.
[0014] The invention also encompasses compositions of matter made
according to the methods disclosed herein.
[0015] Detecting the reaction between the peptide, protein, or
protein complex immobilized on the reaction surface and the reagent
mixture can be accomplished by any means now known in the art or
discovered in the future, including, without limitation, methods
utilizing fluorescent, chemiluminesent, colorimetric, antibody,
and/or radioactive labels. Detecting the reactions can also be
accomplished using mass spectrometry and/or flow cytometry.
[0016] When functionalizing amino-containing groups such as
peptides and proteins to add an acrylamide moiety, it is convenient
to utilize the amino groups at the N-terminus, along with reactive
amino groups present in lysine residues. Thus, peptides and
proteins can be immobilized to the surface at a single point, such
as via a bond formed at the N-terminus. Or multiple bonds may be
formed between the peptide or protein and the surface.
[0017] Moreover, the co-polymerization reaction may proceed using
copolymerizable acrylic and acrylamide moieties that include
linkers capable of being selectively cleaved (e.g., chemically,
enzymatically, or photolytically).
[0018] The present invention can be used to assay any number of
protein interactions, including cleavage, phosphorylation,
methylation, acetylation, hydroxylation, and the like.
BRIEF DESCRIPTION OF THE FIGURES
[0019] FIGS. 1A, 1B, and IC: Formation of protein-acrylamide
hydrogel copolymers on glass surfaces. Fig 1A: Clean glass slides
were treated with (3-acryloxypropyl)-trimethoxysilane, resulting in
the attachment of a surface layer containing the acrylic functional
group. FIG. 1B: Surface-accessible lysine residues and/or the
N-terminus of the protein were reacted with 6-((acryloyl)amino)
hexanoic acid, succinimidyl ester in order to obtain acrylic
functionality on the protein. FIG. 1C: The modified proteins were
then attached to the modified surface through a protein-acrylamide
copolymerization reaction. The .circle-solid. at the far right in
FIGS. 1B and 1C represents the protein. (The structure of the
specific protein used in the Examples is presented in Berman et al.
(2000) Nucleic Acids Res 28:235-42.)
[0020] FIGS. 2A and 2B: Specific vs. nonspecific attachment of
acrylic proteins in polyacrylamide hydrogels. FIG. 2A: Fluorescence
from surface-attached GST-GFP fusion proteins as a function of the
total amount of GST-GFP in the 1 .mu.l spot applied to the surface.
Incorporation of acrylic-labeled GST-GFP into copolymerized
hydrogels was considered specific attachment. Incorporation of
non-acrylic-labeled GST-GFP into copolymerized hydrogels was
considered nonspecific attachment. GFP average gray values are the
mean of four replicates and error bars represent the standard
deviation of the four samples. FIG. 2B: The fluorescence images
that provided the data presented in FIG. 2A. Surface-attached
hydrogel spots are approximately 2 mm in diameter.
[0021] FIG. 3: Linearity of the detection of surface-attached
proteins in polyacrylamide hydrogels. GFP fluorescence values for
the surface attachment of GST-GFP as a function of the total amount
of GST-GFP in the spot applied to the surface. The attachment
chemistry and detection method show considerable linearity in the 3
to 300 ng range. Saturation of the signal above 300 ng is a result
of the fluorescence detection method and not the surface chemistry.
Data for spots containing less than 50 ng of total protein was
acquired with a fluorescence exposure time of 500 ms. Data for
spots containing more than 50 ng of total protein was acquired with
a fluorescence exposure time of 250 ms and multiplied by 1.942 (see
Examples) for comparison with the 500 ms data. Data points are the
mean of four replicates and error bars represent the standard
deviation of the four samples.
[0022] FIGS. 4A and 4B: Lower limits of detection of protein in
polyacrylamide hydrogels. FIG. 4A: Average gray values for
fluorescence and enhanced chemiluminescence detection of
surface-attached GST-GFP. It can be seen that enhanced
chemiluminescence (ECL) detection is more sensitive than detection
based upon GFP fluorescence, allowing the detection of
surface-attached protein in the 0 to 1 ng range. Data points are
the mean of four replicates and error bars represent the standard
deviation of the four samples. FIG. 4B: The chemiluminescence film
that provided the data presented in FIG. 4A.
[0023] FIG. 5: Comparison of ammonium persulfate- (APS) and
ultraviolet light- (UV) induced surface polymerization of protein
into polyacrylamide hydrogels. Fluorescence values for the surface
attachment of GST-GFP as a function the total amount of GST-GFP in
the spot applied to the surface for APS- and UV-induced
polymerization. While UV-induced polymerization results in a higher
level of protein incorporation into the hydrogel, both of the
polymerization and subsequent detection methods resulted in similar
profiles which are linear in the range of 0 to 300 ng. All data
points were obtained with a fluorescence exposure time of 250 ms
and multiplied by 1.942 (see the Examples). Data points are the
mean of four replicates and error bars represent the standard
deviation of the four samples.
[0024] FIGS. 6A and 6B: Diffusion within polyacrylamide hydrogels.
Surface spots (1 .mu.l) of 4%, 10%, and 15% polyacrylamide gel were
equilibrated overnight in a solution containing Texas Red-labeled
dextran (3 kDa). The dextran solution was then replaced with PBST
buffer and the diffusion of dextran from the polyacrylamide
hydrogels was measured as a loss of fluorescence signal. FIG. 6A:
Data shows the fraction of the initial fluorescence signal for
diffusion of 3 kDa dextran from spots of 4%, 10%, and 15%
polyacrylamide. Diffusion of the 3 kDa dextran from the 4% gel
reaches its endpoint within approximately 10 minutes. FIG. 6B:
Pseudocolor representations of the fluorescence intensity of the 4%
polyacrylamide gel spot as a function of time (min:sec). Spot size
is approximately 1.8 mm in diameter.
[0025] FIGS. 7A and 7B: Surface and solution phosphorylation
kinetics. FIG. 7A: ECL values for the detection of phosphorylated
tyrosine on the surface of glass slides as a function the total
amount of GST-EEEIYGEFE (SEQ. ID. NO: 1) in the spot applied to the
surface. Data for phosphorylation reaction times of 10 and 150
minutes is provided. A calculation of the phosphorylated tyrosine
signal based on the Michaelis-Menten parameters is also provided
for the 10 minute reaction time. Data points are the mean of four
replicates and error bars represent the standard deviation of the
four samples. FIG. 7B: Lineweaver-Burk plot for the determination
of surface and solution kinetic values. Solution values:
K.sub.m=2.7.+-.1.0 .mu.M, V.sub.max=8.1.+-.3.1 (arbitrary units).
Surface values: K.sub.m=0.36.+-.0.033 .mu.M, V.sub.max=9.7.+-.0.63
(arbitrary units).
[0026] FIG. 8: Surface phosphorylation kinetics as a function of
polyacrylamide gel percentage. ECL values for the detection of
phosphorylated tyrosine on the surface of glass slides as a
function the total amount of GST-EEEIYGEFE (SEQ. ID. NO: 1) in the
spot applied to the surface after 10 minutes of phosphorylation.
Data points are the mean of six replicates and error bars represent
standard deviation of the six samples. Error bars for 4% and 15%
gels are similar to those shown for the 10% gel.
[0027] FIG. 9: Chemiluminescence film showing detection of v-Abl
tyrosine kinase activity (see Example 9). Surface spots containing
0, 42, 166, 833 and 1660 ng of GST-Crk1 (from left to right) were
phosphorylated by purified v-Abl. Qualitative detection of
phosphorylated tyrosine for the 833 and 1660 ng spots of the two
larger forms of GST-Crkl can be seen.
[0028] FIG. 10: Chemiluminescence film showing detection of Bcr-Abl
tyrosine kinase activity. Surface spots containing 833 ng of
GST-Crkl were printed in duplicate and incubated with a complex
cell lysate containing Bcr-Abl tyrosine kinase. Qualitative
detection of phosphorylated tyrosine for the two longer forms of
GST-Crkl can be seen.
[0029] FIGS. 11A and 11 B: Fluorescence images of GST-GFP spots.
FIG. 11A: Spots containing 0, 95, and 470 .mu.g/ml of GST-GFP were
printed by micropipette and polymerized using UV light.
Spot-to-spot spacing was varied from 500 to 1000 .mu.m using a
Prior microscope stage (Prior Scientific, Inc., Rockland, MA).
Spots containing 95 .mu.g/ml of GST-GFP are approximately 300 .mu.m
in size while spots containing 470 .mu.g/ml of GST-GFP are
approximately 400 .mu.m in size. FIG. 11B: Pseudocolor
representation of the GFP fluorescence data for the spots printed
with 1000 .mu.m spacing. Reproducibility of the spot disposition as
well as distribution of the fluorescence signal throughout the
volume of the spot can be seen.
[0030] FIG. 12: Fluorescence image of surface-attached GST-GFP
spots printed with a spot-to-spot spacing of 250 .mu.m. Spots are
approximately 150 .mu.m in size.
[0031] FIGS. 13A and 13B: Bead-based Abl tyrosine kinase assays.
Anti-phosphotyrosine detection of GST-Crkl (full length), GST-Crkl
(SH3), and GST-Crkl (intra SH3) phosphorylation by v-Abl tyrosine
kinase (FIG. 13A) and K562 cell lysates (FIG. 13B). (+) and (-)
symbols represent the inclusion or exclusion of 100 .mu.M imatinib
in the reaction. The relative positions of GST-Crkl (full length),
GST-Crkl (SH3), and GST-Crkl (intra SH3) are given by markers 1, 2,
and 3, respectively. v-Abl exhibits activity towards GST-Crkl (SH3)
and GST-Crkl (intra SH3) that is inhibited by 100 .mu.M imatinib.
v-Abl phosphorylation of GST-Crkl (full length) is minimal. All
GST-Crkl substrates are phosphorylated by the K562 cell lysate and
lysate activity toward GST-Crkl (full length) and GST-Crkl (SH3) is
inhibited by 100 .mu.M imatinib. Cell lysate activity toward
GST-Crkl (intra SH3) is not inhibited by 100 .mu.M imatinib.
[0032] FIGS. 14A and 14B: v-Abl activity toward Crkl constructs
immobilized in polyacrylamide hydrogels. FIG. 14A: Enhanced
chemiluminescence (ECL) values for the detection of phosphorylated
tyrosine in GST-Crkl (intra SH3) or GST-Crkl (SH3) substrates on
the surface of protein arrays as a function of substrate density.
v-Abl phosphorylation reactions were carried out in the presence of
100 .mu.M ATP. FIG. 14B: Time course data for the phosphorylation
of GST-Crkl (SH3) at 1 h and 2 h reaction times. Protein spots
average 2.54 mm in diameter. Data points are the means of four
replicates, and error bars represent the standard deviation of the
four samples.
[0033] FIG. 15: Imatinib mesylate inhibition of v-Abl activity
toward Crkl constructs immobilized in polyacrylamide hydrogels. ECL
values for the detection of phosphorylated tyrosine in 165
ng/mm.sup.2 spots of GST-Crkl (intra SH3) or GST-Crkl (SH3) as a
function of imatinib concentration. IC.sub.50 for the inhibition of
GST-Crkl (intra SH3) and GST-Crkl (SH3) phosphorylation are 2.0 and
1.5 .mu.M respectively. Protein spots average 2.54 mm in diameter.
Data points are the means of four replicates and error bars
represent the standard deviation of the four samples.
[0034] FIGS. 16A and 16B: K562 cell extract mediated
phosphorylation of Crkl constructs immobilized in polyacrylamide
hydrogels. FIG. 16A: ECL values for the detection of phosphorylated
tyrosine in GST-Crkl (SH3) and GST-Crkl (full length) substrates on
the surface of protein arrays as a function of substrate density.
Bcr-Abl phosphorylation reactions were carried out in the presence
of 10 .mu.M ATP. FIG. 16B: Time course data for the phosphorylation
of GST-Crkl (SH3) at 30 min and 2 h reaction times. Protein spots
average 2.54 mm in diameter. Data points are the means of four
replicates and error bars represent the standard deviation of the
four samples.
[0035] FIG. 17: Imatinib mesylate inhibition of K562-mediated
phosphorylation of Crkl constructs immobilized in polyacrylamide
hydrogels. ECL values for the detection of phosphorylated tyrosine
in 165 ng/mm.sup.2 spots of GST-Crkl (SH3) or GST-Crkl (full
length) as a function of imatinib concentration. IC.sub.50 for the
inhibition of GST-Crkl (SH3) and GST-Crkl (full length)
phosphorylation is 30 .mu.M. Protein spots average 2.54 mm in
diameter. Data points are the means of four replicates and error
bars represent the standard deviation of the four samples.
[0036] FIG. 18: Assessment of phosphatase activity in K562 cell
extracts toward Crkl substrates immobilized in polyacrylamide
hydrogels. Protein arrays containing 165 ng/mm.sup.2 spots of
GST-Crkl (SH3) were phosphorylated for 2 h in the presence of 10
.mu.M ATP and 450 .mu.g of K562 cell lysate. The surfaces were
incubated in the reaction mixture without K562 extract, with K562
extract, and with K562 extract and 300 .mu.M imatinib, representing
a negative control, the potential for phosphatase and Abl kinase
activity, and the potential for only phosphatase activity,
respectively. The level of GST-Crkl phosphorylation is consistent
for reactions up to 2 h in length and decreases only slightly in a
5 h reaction, suggesting minimal GST-Crkl phosphatase activity.
Protein spots average 2.54 mm in diameter. Data points are the
means of four replicates and error bars represent the standard
deviation of the four samples.
[0037] FIG. 19: Detection limit of Bcr-Abl activity for Crkl
constructs immobilized in polyacrylamide hyrogels. Protein arrays
containing 495 ng/mm.sup.2 spots of GST-Crkl (SH3) were incubated
for 2 h in reaction mixtures containing 0 to 100% K562 cell lysate
(Bcr-Abl.sup.+) in a background of HL60 cell lysate (Bcr-Abl-).
Bcr-Abl activity is detectable at 2.5% K562, with statistically
significant results obtains for levels at and above 15% K562 (P
value of 0.02). Protein spots average 2.54 mm in diameter. Data
points are the means of four replicates and error bars represent
the standard deviation of the four samples.
[0038] FIG. 20: Inhibition K562 cell extract-mediated
phosphorylation of Crkl constructs immobilized in polyacrylamide
hydrogels by six different tyrosine kinase inhibitors. Protein
arrays containing 165 ng/mm.sup.2 spots of GST-Crkl (SH3) were
incubated for 2 h in K562 cell lysate reaction mixtures containing
imatinib, PKI166, AG1478, PP2, AG1296, or AG490 at concentrations
ranging from 100 nM to 1 mM. Inhibition of Bcr-Abl activity by
imatinib at concentrations of 10 .mu.M to 1 mM is as expected from
previous data. Significant inhibition of Bcr-Abl activity by PKI
166, AG 1478, and PP2 can be seen at the 1 mM concentration.
Additionally these three inhibitors show slight inhibition at the
100 .mu.M concentration. Protein spots average 2.54 mm in diameter.
Data points are the means of four replicates and error bars
represent the standard deviation of the four samples.
DETAILED DESCRIPTION
[0039] The following abbreviations and definitions are used
throughout the specification and claims:
[0040] APS=ammonium persulfate. Bis
.dbd.N,N'-methylenebisacrylamide. Brij 35 (a registered trademark
of Atlas Chemical Co.)=polyoxyethylene monolauryl ether,
n.about.23. BSA=bovine serum albumin. DMSO=dimethylsulfoxide.
ECL=enhanced chemiluminescence. DTT=dithiothreitol.
EDTA=ethylenediaminetetraacetic acid. GFP=green fluorescent
protein. GST=glutathione S-transferase.
HEPES=N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid.
HRP=horseradish peroxidase.
IPTG=isopropyl-.beta.-D-thiogalactopyranoside.
PBS=phosphate-buffered saline. PBST=phosphate-buffered saline
containing 0.1% Tween-20. PMSF=phenylmethylsulfonyl fluoride (i.e.,
.alpha.-toluenesulfonyl fluoride). SDS=sodium dodecyl sulfate.
STE=salt + Tris/EDTA buffer. TBS=Tris-buffered saline.
TBST=tris-buffered saline containing 0.1% Tween-20.
TEMED=N,N,N',N'-tetramethylethylenediamine.
Tris=tris(hydroxymethyl)aminomethane. Triton X-100 (a registered
trademark of Union Carbide)=p-(1,1,3,3-tetramethylbutyl)phenol
ethoxylate. Tween-20 (a registered trademark of Atlas Chemical
Co.)=polyoxyethylene sorbitan monolaureate, n.about.20.
[0041] The terms "acrylic acid-based," "acrylic-functionalized,"
"acrylamide-based," and "acrylamide-functionalized," denote that
the gel or functionalized reagent includes an acryl moiety,
(CH.sub.2.dbd.CR--C(O)--O)--, or an acrylamide moiety,
(CH.sub.2.dbd.CR--C(O)--NR)--, or a homolog thereof (e.g.,
(alkyl)acrylic acid, (alkyl)acrylamide), etc., where each R is the
same or different from every other R and is selected from the group
consisting of hydrogen; C.sub.1-100 alkyl, alkenyl, or alkynyl;
amino-C.sub.1-100- alkyl, alkenyl, or alkynyl; amido-C.sub.1-100
alkyl, alkenyl, or alkynyl; poly(ethylene glycol).
[0042] The term "polymerizable ethylene-containing monomer unit" is
as defined in U.S. Pat. No. 6,692,912, incorporated herein by
reference. Thus, a polymerizable ethylene-containing monomer unit
is one which, under appropriate reactions conditions, is capable of
copolymerizing with a second polymerizable ethylene-containing
monomer unit. The term explicitly encompasses acryl and acrylamide
moieties. The term explicitly includes monosubstituted ethylenes of
general structure CH.sub.2.dbd.CHX, and unsymmetrical
1,1-disubstituted ethylenes of general structure CH.sub.2.dbd.CXY,
where X and Y are the same or different and are selected from the
group consisting of hydrogen, halo, alkyl, amino, alkylamino,
carboxy, carboxyamido, ethers thereof, and esters thereof. In
preferred embodiments of the invention, the polymerizable
ethylene-containing monomer units are acrylamide, methacrylamide,
acrylic acid, methacrylic acid, derivatives thereof, and esters
thereof. In general, these monomer units are widely available
commercially and are easy to react using standard chemical
procedures. Most are water soluble. See, for example, Sandler &
Karo, "Polymer Synthesis," volume 1, chapters 10 and 12, Academic
Press, Inc., New York, N.Y.(.COPYRGT. 1992); and Sandler &
Karo, "Polymer Synthesis," volume 2, chapter 9, Academic Press,
Inc., New York, N.Y. (.COPYRGT. 1994).
[0043] As used herein, the term "surface" denotes a surface of any
material that does not interfere (or which can be made to be
non-interfering) with the chemistries described herein. A "surface"
can of any geometry, arbitrary or otherwise, including (without
limitation) flat surfaces, beads, flexible films, sheets, blocks,
capillaries, filters, etc. Silica glass surfaces are preferred.
[0044] In the present invention, modified, peptides, proteins, or
protein-containing complexes (collectively referred to hereinafter
as "proteins") are specifically immobilized to a surface by
incorporating the proteins into (or onto) a gel that is itself
immobilized onto the surface. By incorporating the proteins into or
onto a surface-bound gel, the proteins can be immobilized into
addressed arrays on the surface and the concentration of the
proteins within each gel domain can be carefully controlled. This
enables any number of useful studies to be conducted by contacting
the surface with reagent mixtures and examining the resulting
reactions (if any). Thus, surfaces prepared according to the
present invention are useful for conducting, for example, kinetic
studies of enzymes, substrate specificity studies, cross-reactivity
studies, and the like. See the Examples for several different
protocols.
[0045] The general approach calls for copolymerizing a protein
modified to include a polymerizable moiety, with a surface that has
been correspondingly modified to include a another polymerizable
moiety. Thus, a surface, such as a glass slide, a silicon chip, or
other surface is modified to have immobilized thereon at least one
domain comprising a first polymerizable ethylene-containing moiety.
The peptide, protein, or protein-containing complex to be
immobilized onto the surface is then functionalized to include a
second polymerizable ethylene-containing moiety. The functionalized
peptide, protein, or protein-containing complex is then
copolymerized with the domain immobilized on the surface, such that
the peptide, protein, or protein-containing complex is immobilized
upon or within the domain. By controlling the amount of
functionalized protein exposed to the domain during
copolymerization, the concentration of functionalized protein
ultimately immobilized onto the surface can be regulated.
[0046] In the preferred embodiment, the surface has immobilized
thereon at least one domain comprising a gel selected from the
group consisting of acrylic acid-, alkylacrylic acid-, acrylamide-,
and alkylacrylamide-based gels; and the target protein to be
immobilized is modified to include polymerizable acrylic,
alkylacrylic, acrylamide- or alkylacrylamide moiety. The nature of
the peptide, protein, or protein-containing complex to be
immobilized is not critical to the operability or functionality of
the invention. In short, any peptide, protein, or
protein-containing complex, without limitation, can be immobilized
and assayed according to the present invention. In the preferred
embodiment of the invention, the peptide, protein, or
protein-containing complex is an enzyme or a fragment thereof, an
enzyme substrate or a fragment thereof, or a fusion protein or a
fragment thereof. It is much preferred that the functionalized
peptide, protein, or protein-containing is immobilized upon or
within the domain such that the functionalized peptide, protein, or
protein-containing complex remains accessible to participate in
chemical and/or enzymatic reactions. In this fashion, the
reactivity (or other parameters) of the immobilized protein can be
assayed by contacting the surface with suitable reagents and
detecting whether any reaction takes place. In short, the reactive
surface is contacted with a reagent mixture to be assayed and
detecting whether the functionalized peptide, protein, or
protein-containing complex within the reactive surface reacts with
the reagent mixture. See the Examples for specific reactions.
[0047] The functionalized peptide, protein, or protein-containing
complex can be polymerized with the domain immobilized on the
surface using any type of polymerization protocol now known or
developed in the future. Preferred are UV-induced polymerization
and chemical-induced free-radical polymerization. The preferred
initiator for free-radical polymerization is APS. A large number of
free-radical initiators are commercially available and can also be
used in the present invention with equal success, e.g., VAZO-brand
free radical initiators (substituted azonitriles) (DuPont,
Wilmington, Del.). See also Denizov et al. "The Handbook of Free
Radical Initiators," Wiley Press, Hoboken, NJ, .COPYRGT. 2003,
ISBN: 0-471-20753-5.
[0048] A particularly preferred embodiment of the invention
includes attaching peptides, proteins, or protein-containing
complexes to the surface (as described above and in the Examples)
and then assaying the presence, the activity, the chemical
reactivity, or any other physical or chemical characteristic of the
immobilized peptide, protein, and/or protein-containing complex.
This can be accomplished by any number of well known approaches,
including fluorescent, chemiluminesent, calorimetric, antibody, and
radioactive labeling, mass spectrometry, flow cytometry, and the
like. For example, if a kinase substrate is immobilized on the
surface, contacting the surface with a kinase in the presence of
.sup.32P-labeled phosphate will introduce radioactive phosphate
groups into the substrates. The rate and extent of phosphorylation
can then be tracked using a scintillation counter.
EXAMPLES
[0049] The following Examples are included solely to provide a more
complete description of the invention disclosed and claimed herein.
The Examples do not limit the scope of the claims in any
fashion.
Example 1
Preparation of Glass Surface
[0050] Glass microscope slides (Fisher Scientific, Hanover Park,
IL) were cleaned in a 70/30 (v/v) mixture of H.sub.2SO.sub.4/30%
H.sub.2O.sub.2 at 95.degree. C. for 30 minutes. The slides were
thoroughly washed with Milli-Q water (Millipore, Billerica, Mass.)
and dried overnight at 140.degree. C. To deposit the silane
coupling agent on the surface, a 95/5 (v/v) solution of
ethanol/Milli-Q water was prepared and brought to pH 5.0 with
acetic acid. To the ethanol/water solution was added
(3-acryloxypropyl)-trimethoxysilane (Gelest, Tullytown, PA) with
stirring until a final concentration of 2% was reached. Clean glass
slides were immersed in this solution for two minutes, rinsed by
briefly dipping into ethanol, and dried overnight at room
conditions. The slides were then stored in a desiccator at room
temperature until needed.
Example 2
Preparation of Fusion Proteins
[0051] The DNA sequence encoding a mutant green fluorescent protein
(GFP- S65T) from Aequorea victoria was amplified by PCR with
primers containing BamH1 restriction sites flanking the GFP gene
and cloned into the BamH1 restriction site of the pGEX-4T-1 vector
(Amersham Biosciences, Piscataway, N.J.). This plasmid was
transformed into E. coli DH5a cells and the correct insertion of
the GFP gene was confirmed by the isolation of colonies capable of
producing the fluorescent fusion product. (Vectors containing the
GFP- S65T insert are also available commercially from BD
Biosciences, San Jose, Calif.; see also GenBank Accession No.
U36202.)
[0052] For production of the GST-GFP fusion protein, the cells were
grown to mid-log phase at 37.degree. C. in 2.times.YT medium (16 g
tryptone, 10 g yeast extract, 5 g NaCl, pH 6.6 in 1 (one) liter of
water). The culture was then cooled to 30.degree. C. and protein
expression was induced by addition of
isopropyl-.beta.-D-thiogalactopyranoside (IPTG) to a final
concentration of 1 mM. Cells were harvested, washed in PBS (140 mM
NaCl 2.7 mM KCl, 10 mM Na.sub.2HPO.sub.4, 1.7 mM KH.sub.2PO.sub.4,
pH 7.4) and lysed in STE (100 mM NaCl, 10 mM Tris-HCl, 1 mM EDTA,
pH 8.0) containing 1% Triton X-100 and 1 mM PMSF. Cell lysate was
purified by affinity chromatography on a glutathione Sepharose
column following the manufacturer's instructions (Amersham
Biosciences). Briefly, after adding cell lysate to the column, the
column was washed with PBS, pH 7.3 until the eluate was
protein-free. The GST-GFP fusion protein was then eluted by
addition of 50 mM Tris-HCl, pH 8.0 containing 10 mM reduced
glutathione. Purified protein was concentrated in a centrifugal
filter (Millipore) with a 10 kDa nominal molecular weight
cutoff.
[0053] The GST-peptide fusion, GST-EEEIYGEFE (SEQ. ID. NO: 1), was
also expressed and purified in the pGEX-4T-1 vector system. The
consensus sequence for c-Src tyrosine phosphorylation [18] was
expressed as a DNA sequence with an upstream BamH1 site and a
downstream EcoR1 site (restriction sites shown underlined),
5'-AAAAAAA GGATCC GAA GAA GAA ATT TAT GGG GAA TTC GAA GAATTC
CCCCCCC-3' (SEQ. ID. NO: 2). This sequence was doubly digested and
cloned into the BamH1 and EcoR1 restriction sites of the pGEX-4T-1
vector. Correct insertion was verified through DNA sequencing. The
plasmid was transformed into E. coli DH5.alpha. cells, and the
resulting fusion protein was expressed and purified as for the case
of GST-GFP.
Example 3
Labeling of Fusion Proteins
[0054] Purified fusion proteins were transferred to 100 mM sodium
bicarbonate, pH 8.3 buffer and brought to a concentration of
approximately 3 mg/ml through centrifugal concentration. Protein
concentration was measured with a Pierce BCA protein assay kit
(Pierce, Rockford, Ill.). Fusion proteins were then labeled with
6-((acryloyl)amino) hexanoic acid, succinimidyl ester (i.e.,
succinimidyl-6-[(acryloyl)amino] hexanoate) (Molecular Probes,
Eugene, Oreg., catalog no. A20770) according to the manufacture's
directions. Briefly, 100 .mu.l of a 10 mg/ml solution of
6-((acryloyl)amino) hexanoic acid, succinimidyl ester in DMSO was
slowly added to 1 ml of the fusion protein. The reaction was
allowed to proceed for one hour at room conditions with maximum
stirring. The reaction was then quenched by adding 100 .mu.l of
freshly prepared 1.5 M hydroxylamine, pH 8.5. The quenching
reaction was allowed to proceed for one hour at room conditions.
The acrylic-labeled fusion protein was then purified on a G-25
microspin Sephadex column (Amersham Biosciences). Following
purification, labeled fusion proteins were stored at -80.degree. C.
until further use.
Example 4
Attachment of Fusion Proteins to Surfaces
[0055] APS Polymerization: 4% acrylamide solutions for surface
attachment were prepared as follows: [0056] 12.5 .mu.l of 1.5 M
Tris, pH 8.8; [0057] 6 .mu.l of 33% acrylamide mix (0.86 g Bis and
32.14 g acrylamide in a total volume of 100 ml); [0058] 1 .mu.l of
10% APS; [0059] 15 .mu.l of 50% glycerol; [0060] 0.2 .mu.l TEMED;
[0061] 0 to 15 .mu.l acrylic-labeled fusion protein solution; and
water to a total volume of 50 .mu.l.
[0062] After thorough mixing, 1 .mu.l spots were placed onto
acrylic-functionalized slides by pipet and allowed to polymerize
for one hour at room conditions. Slides were then washed by briefly
dipping into approximately 250 ml PBST, followed by a 15-minute
wash and two 5-minute washes with slight agitation in approximately
20 ml of PBST.
[0063] In the case of 10%, and 15% polyacrylamide gels, the
procedure given above was slightly modified to accommodate larger
volumes of the 33% acrylamide mix; 15 .mu.l and 22.7 .mu.l of 33%
acrylamide mix were required for the 10% and 15% gels respectively,
and 7.5 .mu.l of 100% glycerol was substituted for 50%
glycerol.
[0064] UV Polymerization: 4% polyacrylamide solutions for surface
attachment were prepared as follows: [0065] 12.5 .mu.l of 1.5 M
Tris, pH 8.8; [0066] 6 .mu.l of 33% acrylamide mix; [0067] 15 .mu.l
of 50% glycerol; [0068] 0.7 .mu.l TEMED; [0069] 0.125 .mu.l of
freshly prepared 1% methylene blue solution; [0070] 0 to 15 .mu.l
acrylic-labeled fusion protein solution; and water to a total
volume of 50 .mu.l.
[0071] After thorough mixing, 1 .mu.l spots were placed onto
acrylic-functionalized slides by pipet and placed in a UV oven
(Spectronics, Westbury, NY). Polymerization was induced by
illumination with UV light (254 nm) at 1500 .mu.W/cm.sup.2 for 7 to
10 minutes. Slides were then washed as in the case of APS
polymerization. UV polymerization was based on the methylene blue
polymerization strategy of Vasiliskov et al. [19].
Example 5
Diffusion Studies
[0072] Surfaces containing 4%, 10%, and 15%, polyacrylamide gel
spots were prepared as above and then incubated overnight at
4.degree. C. in a PBST solution containing 10 .mu.g/ml of Texas
Red-labeled 3 kDa dextran or 20 .mu.g/ml of Texas Red-labeled 70
kDa dextran (Molecular Probes). The dextran solution was then
replaced with fresh PBST and fluorescence images were taken over
time to assess diffusion from the surface spots.
Example 6
Solution Kinase Assays
[0073] Solution protein kinase assays with protein-tyrosine kinase
p.sub.60.sup.c-src (Calbiochem, San Diego, Calif.) were carried out
according to the manufacture's instructions. Briefly, reaction
mixtures containing 1.5 .mu.l of 50% glycerol, 1.5 .mu.l of ATP mix
(0.15 mM ATP, 30 mM MgCl.sub.2), 0.3 units of p60.sup.c-src in 1.5
.mu.l kinase dilution buffer (50 mM HEPES, 0.1 mM EDTA, 0.015% Brij
35, 0.1 mg/ml BSA, 0.2% P-mercaptoethanol, pH 7.5), and 1 .mu.l
GST-EEEIYGEFE (SEQ. ID. NO: 1) solution (50 mM HEPES, 0.1 mM EDTA,
0.015% Brij 35, pH 7.5 containing anywhere from 0 to 600 ng of
GST-EEEIYGEFE) were incubated at 37.degree. C. for times ranging
from 10 minutes to 5.5 hours. After each kinase reaction the
samples were mixed with 94.5 .mu.l of TBS (10 mM Tris-HCl, 100 mM
NaCl, pH 7.5) containing 1% SDS and heated to 97.degree. C. for 5
minutes. Upon cooling, the samples were transferred to a
nitrocellulose membrane using a Dot Blot apparatus (Biorad,
Hercules, Calif.) following the manufacture's instructions. Prior
to assembly of the Dot Blot apparatus, the membrane was wetted in
TBS for 10 minutes. The apparatus was then assembled and each well
was washed twice with 100 .mu.l of TBS. 50 .mu.l of the kinase
reaction product was applied to each well. After application of the
reaction mixture the wells were washed twice with 200 .mu.l of TBS
containing 0.05% Tween-20.
Example 7
Surface Kinase Assays
[0074] Surface protein kinase assays were carried out in a fashion
similar to the solution kinase assays. Glass slides containing 0 to
600 ng of GST-EEEIYGEFE (SEQ. ID. NO: 1) per 1 .mu.l polyacrylamide
spot were prepared as above and stored overnight in kinase assay
buffer (50 mM HEPES, 0.1 mM EDTA, 0.015% Brij 35, pH 7.5) at
4.degree. C. Just prior to the kinase assay, these slides were
dried under a stream of compressed air. Careful attention was paid
to make sure that only the bare portions of the slide were dried
and the polyacrylamide spots remained hydrated. The protein kinase
reaction mixture containing 1.5 .mu.l of 50% glycerol, 1.5 .mu.l of
ATP mix, and 0.3 units of p60.sup.c-src in 1.5 .mu.l of kinase
dilution buffer was placed on top of each polyacrylamide spot and
the slides were incubated in a humidity chamber at 37.degree. C.
for 10 minutes to 5.5 hours. After the reaction, each slide was
washed by briefly dipping into approximately 250 ml TBST followed
by a 15-minute wash and two 5-minute washes (with slight agitation)
in approximately 20 ml of TBST.
Example 8
Fluorescence and Chemiluminescence Detection of Surface-Attached
and Phosphorylated Proteins
[0075] GFP and Texas Red Detection. Quantitative GFP and Texas Red
fluorescence measurements were obtained using an inverted
epifluorescence Olympus microscope (Olympus, Melville, NY) coupled
to a Spot CCD camera (Diagnostic Instruments, Sterling Heights, MI)
and MetaVue image acquisition and analysis software (Universal
Imaging Corporation, Downingtown, PA). Images were taken with a
4.times.Olympus objective and exposure times of 10 ms for diffusion
studies and 250 or 500 ms for GFP analysis. Exposure times were
chosen so that the fluorescent signal remained in the linear
operating range of the camera. The fluorescent signal from each
surface spot was corrected for background by subtracting the
background signal in the near vicinity of the spot. After
subtraction of background, signals from 250 ms GFP exposures were
multiplied by 1.942 for comparison with data obtained from 500 ms
exposures. This conversion factor was determined by comparing the
fluorescent signals from duplicate 250 ms and 500 ms exposures of
spots containing 0, 14.4, 28.8, 57.6, and 172.7 ng of GST-GFP. The
resulting relationship between 250 ms and 500 ms exposure data was
found to be independent of signal intensity within the linear
operating range of the camera. Diffusion study controls exhibited
insignificant loss of fluorescent signal due to bleaching.
[0076] Chemiluminescence Antibody Detection. In the case of GST-GFP
detection, slides were blocked in approximately 20 ml of PBST
containing 5% milk for 1 hour at room conditions. Each slide was
then removed from the blocking solution and incubated with 3 .mu.g
of anti-GST antibody (Molecular Probes) in 1.5 ml of PBST
containing 5% milk for 1 hour. Each slide was then washed by
briefly dipping into approximately 250 ml PBST followed by a
15-minute wash and two 5-minute washes (with slight agitation) in
approximately 20 ml of PBST. Each slide was then incubated with
0.75 .mu.g of HRP-conjugated secondary antibody (Molecular Probes)
in 1.5 ml of PBST containing 5% milk for 1 hour and subsequently
washed in PBST as above. The slides were then stored in PBST until
detection.
[0077] In the case of phosphotyrosine detection, a similar
procedure was followed with the substitution of TBST for PBST as
the buffer and BSA for milk as the blocking agent. These
substitutions were required to reduce the nonspecific binding of
the secondary antibody used in this detection scheme. Slides were
blocked in approximately 20 ml of TBST containing 1% BSA for 1 hour
at room conditions. Each slide was then removed from the blocking
solution and incubated with 1.5 .mu.g of monoclonal
anti-phosphotyrosine antibody (Sigma, Saint Louis, Mo.) in 1.5 ml
of TBST for 1 hour. Each slide was then washed by briefly dipping
into approximately 250 ml TBST followed by a 15-minute wash and two
5-minute washes (with slight agitation) in approximately 20 ml of
TBST. Each slide was then incubated with 0.15 .mu.g of
HRP-conjugated secondary antibody (Molecular Probes) in 1.5 ml of
TBST for 1 hour and subsequently washed in TBST as above. The
slides were then stored in TBST until detection.
[0078] Nitrocellulose membranes from the solution kinase studies
were treated in a manner analogous to that used for the slides. In
this case, the antibody incubations consisted of 25 .mu.g of
monoclonal anti-phosphotyrosine antibody in 25 ml of TBST and 2.5
.mu.g of HRP-conjugated secondary antibody in 25 ml of TBST.
[0079] Enhanced chemiluminescence (ECL) western blotting detection
reagent (Amersham Biosciences) was used to detect HRP-labeled
slides and membranes. Briefly, after removal from the wash buffer,
each slide was treated with 1.5 ml of ECL detection reagent for 1
minute. In the case of the nitrocellulose membranes, 20 ml of ECL
detection reagent was used per membrane. The detection reagent was
then "shaken off" from the slide and the slide was placed between
transparent acetate sheets for subsequent exposure to film
(Amersham Biosciences). After development, the average gray value
of each spot was obtained using ImageJ software (NIH, Bethesda,
Md.).
Example 9
Surface-Based Detection of v-Abl and Bcr-Abl Kinase Activity
[0080] Protein kinases are a fundamental component of cellular
signaling pathways and are involved in a wide variety of cellular
responses including growth, proliferation, differentiation, and
migration. These diverse roles are carried out through the protein
kinase-catalyzed transfer of phosphate groups from ATP to tyrosine
or serine/threonine residues within specific substrates. Tyrosine
phosphorylation by protein tyrosine kinases (PTKs) is particularly
important, resulting in the formation of new binding sites for
proteins containing motifs specific to various signaling and
regulatory pathways. Normally, PTKs are maintained in a low
activity state through autoregulatory mechanisms involving the PTK
enzymes themselves and cellular tyrosine phosphatases. Thus, less
than 1% of cellular PTKs are in the active state [27]. Upon binding
a ligand or other protein, PTKs become activated and can
subsequently phosphorylate other cell-signaling components before
returning to the inactive state [28-31].
[0081] Protein tyrosine kinases with altered activity have been
implicated in a variety of diseases including cancer. Specific PTKs
identified as oncogenic include EGFR, PDGFR, FGFR, VEGF, Her-2/neu,
Bcr-Abl, Tel-Abl, c-Kit, and c-Met [28, 30-33]. These oncogenes
affect numerous pathways critical to cancer cell survival and
progression, including cell cycle regulation, apoptosis, and
genetic repair mechanisms [30]. As a result of their function in
numerous types of cancer, PTKs have become the focus of active
fundamental biochemical research efforts. They are also potential
targets for rationally designed pharmaceutical kinase inhibitors
[28, 30, 32].
[0082] Chronic Myeloid Leukemia (CML) is a cancer of the blood
cells which results in the replacement of the hematopoietic cells
in the bone marrow with leukemic cells. CML comprises about 20% of
all leukemia cases, affecting approximately 1 in every 100,000
individuals [34]. For a vast majority of patients, CML is caused by
a reciprocal translocation between the long arms of chromosomes 9
and 22, termed the Philadelphia chromosome [35]. The result is the
fusion of two genes (BCR from chromosome 22 and ABL from chromosome
9) to create the fusion gene BCR-ABL. This gene is expressed to
give the Bcr-Abl fusion protein, a constitutively active version of
the normal cellular protein tyrosine kinase c-Abl. Thus, because
Bcr-Abl is constitutively active, there is a loss of the regulation
of cellular signaling pathways involving c-Abl. The end result of
Bcr-Abl expression is increased cellular proliferation, reduced
apoptosis, disturbed cellular adhesion and migration, genetic
instability, and a massive myeloid expansion (the clinical hallmark
of CML) [27, 34].
[0083] Three therapies are currently available for the treatment of
CML: alpha-interferon treatment, bone marrow transplantation, and
tyrosine kinase inhibition with Glivec.TM.. Treatment with
.alpha.-interferon has been shown to prolong survival by about a
year as compared to treatment with conventional chemotherapy. In
addition, approximately 6 to 20% of patients show a complete
cytogenetic remission (no Philadelphia chromosomes detectable).
Alpha-interferon treatment is often associated with significant
side effects, making prolonged treatment intolerable in many
instances.
[0084] Bone marrow transplantation is the only curative CML
treatment and results in the long-term, leukemia-free survival of
50 to 80% of patients. However, this option is only available to
patients who have a suitable donor and can withstand the treatment.
Thus, bone marrow transplantation is limited to approximately 25%
of CML patients [27, 34, 36].
[0085] Approved by the FDA in late 2002, Glivec is a tyrosine
kinase inhibitor that specifically targets Bcr-Abl. Clinical trials
have shown that Glivec treatment results in complete cytogenetic
remission in 41% of patients and that treatment is associated with
much milder side effects than .alpha.-interferon treatment or bone
marrow transplantation [27, 34]. Despite these promising results,
CML patients, especially those with advanced stages of the
leukemia, have shown resistance to Glivec treatment and many have
relapsed within several months [27, 34, 37]. Molecular biological
techniques have revealed that BCR-ABL gene amplification and a
variety of Bcr-Abl point mutations are the cause of this clinical
resistance [38].
[0086] Currently there is an ongoing debate as to the most
effective course of treatment for CML. Doctors could start with
.alpha.-interferon and then move to Glivec and eventually bone
marrow transplantation if treatment is not progressing
satisfactorily [37]. Alternatively, harsh chemotherapeutics could
be used initially followed by Glivec or a treatment based solely on
Glivec could be used [34]. Despite this debate, the continued
monitoring of CML patients and their response to treatment is a
commonality in the area [27]. While mutational analysis and western
blotting [36] have been used to study Bcr-Abl resistance to
treatment, a fast, efficient, quantitative, and reliable system for
clinical use has yet to be developed. The present invention, as
shown by this Example, provides such a diagnostic system.
[0087] Fusion Protein: Three GST-Crkl fusion proteins, GST-Crkl
(full length), GST-Crkl (both SH-3's), and GST-Crkl (intra SH3)
were obtained from Dr. Brian Druker and expressed and purified as
for the case of GST-GFP [see Example 2 and reference 39]. The CRKL
protein was identified as a substrate for the BCR-ABL tyrosine
kinase in patients with chronic myelogenous leukemia. Senechal et
al. [40] reported that CRKL is phosphorylated when overexpressed,
activates RAS and JUN kinase signaling pathways, and transforms
fibroblasts in a RAS-dependent fashion.
[0088] Source of Bcr-Abl tyrosine kinase: K562 cells (American Type
Culture Collection, Manassas, Va.) were grown in RPMI-1640 media
containing L-glutamine supplemented with 10% fetal bovine serum,
100 units/ml penicillin, and 100 pg/ml streptomycin (Cambrex Bio
Science, Walkersville, MD) at 37.degree. C. in 5% CO.sub.2. The
lysis buffer contained 42.275 mM HEPES, pH 7.3, 126.82 mM NaCl,
1.268 mM MgCl.sub.2, 0.846 mM EDTA, 84.6 mM NaF, 8.46 mM sodium
pyrophosphate, 0.169 sodium orthovanadate, 1 mM PMSF, 4% complete
protease inhibitor (v/v) (Roche, Indianapolis, Ind.), 0.95% Triton
X-100 (v/v), and 9.5% glycerol (v/v). Cells were spun down,
resuspended at a concentration of 1.times.10.sup.6 cells/ml in the
lysis buffer, and incubated on ice for 20 min. Cell lysate was then
obtained by spinning at 1,500 rpm in a microcentrifuge for 10 min
and stored at -80.degree. C. until subsequent use.
[0089] Surface protein kinase assays: Surface protein kinase assays
were carried out in a fashion similar to that for the c-Src surface
protein kinase assays described in Example 7. Glass slides
containing 0 to 1660 ng of the of GST-Crkl fusion proteins per 1
.mu.l polyacrylamide spot were prepared as above (see Examples 1-4)
and stored overnight in Abl kinase assay buffer (50 mM Tris-HCl, 10
mM MgCl.sub.2, 0.1 mM EDTA, 1 mM DTT, 0.015% Brij 35, 100 .mu.g/ml
BSA, pH 7.5) at 4.degree. C. Just prior to the kinase assay, these
slides were dried under a stream of compressed air. Careful
attention was paid to make sure that only the bare portions of the
slide were dried and the polyacrylamide spots remained
hydrated.
[0090] The v-Abl protein kinase reaction mixture containing 1.5
.mu.l of 50% glycerol, 100 .mu.M ATP, 1.5 .mu.L 3.times.Abl kinase
assay buffer, and 10 units of v-Abl (Calbiochem) in 1.5 .mu.l of
water was placed on top of each polyacrylamide spot and the slides
were incubated in a humidity chamber at 37.degree. C. for 2 hours.
The Bcr-Abl kinase reaction mixture containing 25 .mu.l of 50%
glycerol, 100 .mu.M ATP, 25 .mu.L 3.times.Abl kinase assay buffer,
and 18.2 .mu.g of K562 cell lysate in 25 .mu.l of water was placed
on top of the polyacrylamide spot array and the slides were
incubated in a humidity chamber at 37.degree. C. for 2 hours. After
the reaction, each slide was washed by briefly dipping into
approximately 250 ml TBST followed by a 15-minute wash and two
5-minute washes (with slight agitation) in approximately 20 ml of
TBST.
[0091] Detection of Phosphorylation: The extent of phosphorylation
was determined using the same methodology described in Example
8.
Example 10
Printing of "Microarray" Spots
[0092] The GST-GFP fusion protein as described in Example 2 was
used to fabricate the microarray. The fusion protein was attached
to the surface as described in Example 4, with slight
modification:
[0093] UV Polymerization: 4% polyacrylamide solutions for surface
attachment were prepared as follows: [0094] 12.5 .mu.l of 1.5 M
Tris, pH 8.8; [0095] 6 .mu.l of 33% acrylamide mix; [0096] 15 .mu.l
of 50% glycerol; [0097] 0.7 .mu.l TEMED; [0098] 0.75 .mu.l of
freshly prepared 1% methylene blue solution; [0099] 0, 1, or 5
.mu.l acrylic-labeled fusion protein solution; and water to a total
volume of 50 .mu.l.
[0100] After thorough mixing, spots were placed onto
acrylic-functionalized slides by initiating fluid contact between a
micropipette and an acrylic functionalized glass slide. The pattern
of spot deposition was controlled using the x-axis and y-axis
motion of a Prior microscope stage (Prior Scientific, Rockland,
MD). Printed slides were then placed in a UV oven (Spectronics,
Westbury, NY) and polymerization was induced by illumination with
UV light (254 nm) at 1500 .mu.W/cm.sup.2 for 10 minutes in a
nitrogen environment. Slides were then washed as in the case of APS
polymerization (see Example 4).
[0101] Fluorescence from the GFP was detected as in Example 8.
Results are shown in FIG. 11. FIG. 12 depicts a fluorescence image
of surface-attached GST-GFP domains printed with a domain-to-domain
spacing of about 250 .mu.m.; each domain is approximately 150 .mu.m
in size. Results and Significance of Example 1-10:
[0102] The reactions used to incorporate the GST-fusion protein
into a polyacrylamide gel, and then linking the gel to an acrylated
glass surface are depicted in FIGS. 1A, 1B, and IC. Initially,
clean glass slides were treated with
(3-acryloxypropyl)-trimethoxysilane resulting in the formation of
an acrylic- functionalized glass surface (FIG. 1A). An acrylic
functional group is also attached to purified fusion proteins by
use of a succinimidyl ester, as shown in FIG. 1B. The ester reacts
with the N-terminal amino group and .epsilon.-amino group of lysine
residues contained within the protein. In the Examples, GST-GFP and
GST-EEEIYGEFE (SEQ. ID. NO: 1) fusion proteins were used as model
proteins. GST-GFP presents 21 surface-accessible lysine residues
and GST-EEEIYGEFE presents 9 surface-accessible lysine residues
[20].
[0103] Following functionalization of the surface and the fusion
proteins with acrylic groups, the proteins were attached to the
surface through the acrylamide copolymerization scheme shown in Fig
IC. Surface-attached GST-GFP incorporated in the polyacrylamide
hydrogel maintained its fluorescence for several weeks at room
temperature and several months when stored at 4.degree. C., clearly
demonstrating a strong and stable surface attachment of the GST-GRP
(data not shown). Data from GST-GFP attachment also showed that the
copolymerization reaction was fairly efficient at immobilizing
acrylic-labeled GST-GFP, with an average incorporation within the
gel spot of approximately 60% of the GST-GFP applied to the
surface. The incorporation efficiency was independent of protein
concentration within the range studied (data not shown).
[0104] Several tests were performed on arrays of immobilized
GST-GFP to determine the characteristics of the attachment method.
Initially, studies investigated the amount of specific vs.
nonspecific surface attachment of protein. In these studies the
amount of protein contained within the 1 .mu.l spot prior to
copolymerization on the surface of a glass slide was varied from 0
to 30 ng. Surface attachment of acrylic-labeled proteins to
acrylic-functionalized glass surfaces was considered to be the sum
of the specific and nonspecific surface attachment. Attachment of
acrylic-labeled proteins to non-acrylic-functionalized glass
surfaces or attachment of non-acrylic-labeled proteins to
acrylic-functionalized glass surfaces were considered to be
nonspecific attachment. It can be seen from FIGS. 2A and 2B that
the specific attachment of acrylic-labeled GST-GFP fusion proteins
to acrylic-functionalized glass surfaces is at least 7-fold greater
than the nonspecific attachment of non-acrylic labeled GST-GFP to
the same surface over the concentration range studied. In FIG. 2A,
the specific attachment is designated by (.diamond-solid.) and
non-specific attachment by (.DELTA.). FIG. 2B depicts the gel from
which the date in FIG. 2A was generated. Nonspecific attachment of
acrylic-labeled GST-GFP to clean glass surfaces was minimal (data
not shown).
[0105] Having shown the specificity of the surface attachment
strategy, a series of Examples was then performed to determine the
overall concentration-dependence of the attachment and detection
method, as well as the upper and lower detection limits. In these
Examples the amount of GST-GFP contained within the 1 .mu.l spot
prior to copolymerization on the surface of an acrylated glass
slide was varied from 0 to 600 ng. FIG. 3 demonstrates that the
attachment of GST-GFP provides a detectable GFP fluorescence signal
at levels equal to and above about 1 ng. Given the average spot
diameter of 2 mm, this corresponds to a lower GFP detection limit
of about 0.32 ng/mm.sup.2. The polyacrylamide surface attachment
strategy also provides for linear detection of GST-GFP in the range
of from at least about 3 to about 300 ng of protein. It must be
noted, however, that the saturation of the signal between 300 and
600 ng in the Example is an artifact of the fluorescence detection
method, and not a limitation inherent to the attachment strategy.
For instance, by adjusting the acquisition time on the CCD camera
attached to the fluorescence microscope, the linearity of the
attachment can be extended well beyond the 300 ng mark shown in
FIG. 3 (data not shown).
[0106] The lower detection limit of proteins polymerized within the
protein-acrylamide hydrogels decreased when enzyme-conjugated
antibodies were used to amplify the signal. FIG. 4A shows the
fluorescent and chemiluminescent signals from 1 .mu.l spots
containing 0 to 3 ng of GST-GFP. (FIG. 4B shows the gel from which
the data in FIG. 4A were generated.) Chemiluminescence signals were
obtained by labeling the protein with a primary anti-GST antibody
followed by a secondary antibody conjugated to HRP. The data show
that the ECL detection method is more sensitive than the direct GFP
detection method in this range, allowing the detection of protein
concentrations as low as 0.080 ng/mm.sup.2 of surface-attached
GST-GFP. However, this increase in sensitivity comes with an
increase in the variability (standard deviation) of the
measurements, as well as a decrease in the saturation limit of the
detection method as compared to GFP fluorescence detection. Despite
these drawbacks, FIG. 4A shows that ECL carried out under the
conditions described in Example 8 is capable of quantitatively
measuring surface-attached protein in the 0 to about 6 ng range. It
is possible to extend the signal saturation limit by further
dilution of the antibodies, dilution of the ECL detection reagents,
or decreasing the film exposure time (data not shown).
[0107] FIG. 5 provides the results of surface attachment strategies
which rely on two different initiation methods of the
polyacrylamide polymerization reaction; all other results in this
paper have been derived from APS induced polymerization unless
specifically noted. (See Example 4.) Incorporation of GST-GFP into
protein-acrylamide copolymer hydrogels is slightly more efficient
in the case of UV-induced polymerization. However, both
polymerization and subsequent detection methods provide a linear
fluorescence signal in the range of from 0 to about 300 ng protein.
In FIG. 5, APS polymerization is shown by (.diamond-solid.); UV
polymerization by (.quadrature.).
[0108] To characterize protein accessibility within the hydrogel,
the porosity of polyacrylamide hydrogels was measured as a function
of the acrylamide monomer concentration (see FIG. 6A; FIG. 6B
depicts the gel from which the data presented in FIG. 6A were
generated). Diffusion of a fluorescently-labeled 3 kDa dextran from
spots of 4%, 10%, and 15% polyacrylamide gel without immobilized
protein was calculated by measuring the loss of fluorescence as
this dextran diffused from the hydrogel. Time course data given in
FIG. 6A show that diffusion from 4% gel spots is complete within
approximately 10 minutes and occurs significantly faster than
diffusion from 10% or 15% polyacrylamide gel spots. This indicates
a higher porosity in gels containing less acrylamide.
Epifluorescence intensity profiles of the polyacrylamide gel spots
loaded with the 3 kDa dextran show lower fluorescence near the edge
of the gel and higher fluorescence near the middle of the gel spot
(see FIG. 6B). As the diffusion experiments proceed, a loss of the
dextran from the spot was observed, with the intensity of the outer
regions of the image circle decreasing much faster than the
intensity in the middle. This is consistent with an initial uniform
dispersion of the dextran throughout the volume of a hemispherical
spot, followed by diffusion in the radial direction.
[0109] Diffusion of a 70 kDa dextran was also investigated, but no
dextran loading into the surface spots was observed after an
overnight incubation in the dye solution (data not shown). This
observation also illustrates that surface adsorption of the
conjugated dextrans to the polyacrylamide gel spots is negligible.
The present assumption is that the pore size of the polyacrylamide
gels limits unassisted diffusion of molecules of this size into the
gel spots. Inclusion of dextran dyes in the polyacrylamide
polymerization mixture was investigated as an additional method of
gel loading. However, the presence of dextran during polymerization
affects gel formation.
[0110] While measurements of protein concentration are useful,
additional insight into biological systems is often gained from
characterizing protein activity. Therefore, Examples 6 and 7 were
performed to determine the kinetics of a phosphorylation reaction
utilizing a surface-attached substrate. The fusion protein
GST-EEEIYGEFE (SEQ. ID. NO: 1) was acrylic labeled, copolymerized
within 1 .mu.l hydrogel spots, and phosphorylated by c-Src.
Phosphorylation of surface-attached GST-EEEIYGEFE appears to follow
Michaelis-Menten kinetics (FIG. 7A). The kinetics of GST-EEEIYGEFE
(SEQ. ID. NO: 1) phosphorylation by c-Src, in solution and on the
surface, are compared in the form of the Lineweaver-Burk plot given
in FIG. 7B. From these data the kinetic values for the reaction
occurring in solution were determined to be K.sub.m=2.7 +1.0 .mu.M
and V.sub.max=8.1.+-.3.1 (arbitrary units). These values are
similar to values previously reported for solution phosphorylation
of the c-Src consensus sequence AEEEIYGEFEAKKKK (SEQ. ID. NO: 3)
[18].
[0111] Kinetic values for the reaction utilizing
surface-immobilized substrate were K.sub.m=0.36.+-.0.033 .mu.M and
V.sub.max=9.7+0.63 (arbitrary units). It is clear from these data
that although the reactions proceed at similar maximum rates, the
K.sub.m value for the surface reaction is approximately 10-fold
lower than that in solution.
[0112] FIG. 8 shows phosphorylation of surface-attached
GST-EEEIYGEFE (SEQ. ID. NO: 1) as a function of the acrylamide
concentration used for immobilization. The rate of phosphorylation
is not a function of acrylamide concentration, suggesting that the
accessibility of substrate to c-Src, and the accessibility a
phosphorylated substrate to primary and secondary detection
antibodies, are similar in each of the three hydrogels. Given that
c-Src is a 60 kDa protein and the antibodies are approximately 150
kDa in size, this observation is supported by the diffusion data
(which showed a lack of diffusion of a 70 kDa dextran into the gel
spots regardless of gel percentage).
[0113] In short, the present invention comprises a method by which
proteins can be attached to glass surfaces through copolymerization
into a polyacrylamide hydrogel. The reaction between an
acrylic-functionalized glass surface and acrylic-labeled proteins
results in the specific attachment of the protein to the glass
surface, likely involving covalent interactions between the
protein, gel, and surface. Acrylic labeling occurs at primary
amines, including lysine residues on the exterior of the protein or
the N-terminus of the protein. Although this method will not result
in a uniform orientation of the protein in the polyacrylamide
hydrogel, it is applicable to, and will function with, any protein
containing a surface-accessible lysine residue or
surface-accessible N-terminus. Furthermore, attachment of the
protein in several different orientations allows accessibility to
different regions of the protein, a property which is highly useful
in proteomic experiments focusing on (for example and not by way of
limitation): identifying novel protein properties, mapping
epitopes, and evaluating the binding properties of different
epitopes.
[0114] The polyacrylamide attachment strategy disclosed herein is a
distinct improvement compared to reported attachment strategies
based on aldehyde-coated glass slides [8, 9, 12]. In the
aldehyde-based strategies, the protein amino groups are linked
directly to the surface. The present approach, however, uses an
intervening acrylic linking moiety. In short, in the present
invention, the protein amino groups are labeled with an acrylic
functional group. While this added step may require additional time
and cost to produce the protein array, it effectively eliminates
the problem of nonspecific binding of the protein to the surface.
In the prior art aldehyde-based approach, the free aldehyde groups
will react with any protein that comes into contact with the
microarray surface, and therefore must be blocked with BSA before
microarray experiments can be performed. In the present invention,
however, the acrylic-functionalized glass slides will only
polymerize with the acrylic-labeled proteins. Moreover, the
polymerization reaction will occur only when externally initiated.
Thus, the present invention provides far greater flexibility to
control both the fabrication of the microarray itself, as well as
the reactions that are to be conducted using the microarray.
[0115] In addition to the aldehyde reaction strategy, there are
several other attachment strategies that result in covalent surface
attachment of proteins. Protein attachment to alkanethiols on gold
surfaces [6, 21], glyoxylyl-modified glass slides [22], and epoxy
coated slides [10] have been reported. These methods all result in
an attached protein that is relatively close to the surface. In
such proximity to the surface, the protein may interact
nonspecifically with the surface or become inaccessible to large
components in solution above the surface. This phenomenon has been
seen in experiments involving peptides attached to aldehyde
surfaces. These peptides have been sterically obscured from enzymes
in solution by the BSA used in the blocking step and the authors
were required to modify the aldehyde attachment scheme to produce
the desired results [9].
[0116] In contrast, the present invention provides a polyacrylamide
buffer layer between the protein and the surface. This layer serves
to prevent hydrophobic interactions between the surface and
protein, resulting in lower levels of surface denaturation. The
polyacrylamide hydrogel in which the protein is immobilized can
also serve to maintain a microenvironment around the protein which
helps to maintain the protein in a hydrated state.
[0117] In addition to providing a buffer layer and
microenvironment, the polyacrylamide matrix provides a porous,
three-dimensional structure in which proteins can be immobilized.
Thus, the capacity of the surface is greatly increased as compared
to strategies in which the protein is directly attached to the
surface (thereby placing inherent limits on the overall density of
the microarray). Example 5 shows that virtually the entire
surface-attached gel is accessible to small molecules (i.e. those
molecules with molecular weights on the order of a few thousand
Daltons) and that diffusion occurs within a matter of minutes.
[0118] Notably, only a fraction of the 4% polyacrylamide gel was
accessible to molecules in the 60 to 70 kDa size range (see Example
5). Such discrimination is highly useful in proteomic or drug
discovery applications where an immobilized protein-small molecule
interaction is being sought from a complex mixture, e.g., toxicity
associated with drug metabolism [23, 24]. In addition,
well-established polyacrylamide chemistry exists for controlling
gel properties such as pore size and pore size distribution.
[0119] For example, other proteomic applications may benefit from
larger pore sizes and a fully accessible gel spot. Rubina et. al.
have reported increased diffusivity and porosity within gels formed
from methacrylamide [17]. Increases in porosity have also been
reported for gels polymerized with variations on the acrylamide
monomer and cross-linker Bis or changes in the polymerization
conditions [25, 26]. Such modifications can be easily accomplished
in the present surface attachment system simply by changing the
copolymerization mixture.
[0120] Regardless of the polyacrylamide gel pore size, the
diffusivity within the hydrogel will remain lower than that in
solution. This difference, however, decreases proportionately with
the size of the spots. Thus, decreasing the spots from a volume of
1 .mu.l to 1 nanoliter is beneficial to minimize reaction
limitations due to diffusion. To facilitate this transition,
Example 4 demonstrates that UV-induced initiation of the
polymerization reaction results in attachment characteristics
essentially identical to those for APS-induced polymerization. This
result is important in that the attachment strategy described
herein can be scaled up to create microarrays using commercially
available microarray printing devices and subsequent UV-induced
polymerization.
[0121] In Examples 7 and 8, the attachment strategy has been used
to study the c-Src tyrosine phosphorylation of surface-attached
substrates. Michaelis-Menten kinetic values for this system were
K.sub.m=2.7+1.0 .mu.M, V.sub.max=8.1+3.1 (arbitrary units) and
K.sub.m=0.36.+-.0.033 .mu.M, V.sub.max=9.7.+-.0.63 for the solution
and surface reactions respectively. While this study provides
insight into the kinetics of surface phosphorylation vs. solution
phosphorylation, it also serves to demonstrate the utility of the
surface attachment strategy to create microarrays in which a
variety of protein properties can be studied. This includes studies
identifying kinase specificity or inhibition, identifying novel
enzyme substrates, or identifying protein-protein interactions such
as those involved in the formation of cell-signaling complexes. The
combination of the surface attachment strategy and continued
advances in peptide and protein libraries, high-throughput cloning
techniques, and fluorescence- and mass spectrometry-based
detection, results in a powerful and highly useful proteomics tool
with a wide range of applications.
[0122] While Examples 7 and 8 demonstrate that the subject
invention can measure the phosphorylation of surface-attached
proteins by the action of c-Scr, Example 9 further demonstrates
that the subject invention can measure the phosphorylation of
surface-attached GST-Crkl fusion proteins by the kinases v-Abl and
Bcr-Abl. In Example 9, fusion proteins corresponding to three
different lengths of Crkl fused to GST were acrylic labeled and
then copolymerized within 1 .mu.l hydrogel spots. Initially, these
substrates were phosphorylated by purified v-Abl (see FIG. 9).
Surfaces containing GST-Crkl copolymer spots were also incubated
with a K562 cell lysate containing the tyrosine kinase Bcr-Abl (see
FIG. 10).
[0123] Detection of GST-Crkl phosphorylation by purified v-Abl
demonstrates that the invention will work with tyrosine kinases
other then c-Src.
[0124] Far more important, however, is that Example 9 demonstrates
that the present invention can detect GST-Crkl phosphorylation by
Bcr-Abl in a complex cell lysate. This is a tremendous advancement
over conventional assays. The ability to detect the action of a
kinase from a complex cell lysate, without interference from other
enzymes or proteins in the cell lysate, greatly increases the ease
and utility of the invention because extensive purification of the
test mixture is unnecessary. To the inventors' knowledge, Example 9
may be the first report of surface-based detection of protein
activity from a complex mixture such as a cell lysate.
[0125] Thus, Example 9 demonstrates that the present invention can
be used as a simple, reliable, and cost-effective method of
detecting Bcr-Abl activity in extracts from patients with CML. The
invention thus provides oncologists with a direct measure of an
individual patient's response and/or resistance to treatment. This
capability is a vast improvement over the indirect measures used
conventionally (blood cell counts and cytogenetics). Guided by the
results provided by the present invention, an individualized
treatment program can now be undertaken for each patient and
treatment alternatives .alpha.-interferon, early bone marrow
transplant, or other pharmaceutical inhibitors currently in
development [27, 34]) could be explored in patients that exhibit
resistance to Glivec. In short, the efficacy of the initial
anti-CML treatment chosen can be evaluated and that treatment
altered if the results of the present assay indicate that Bcr-Abl
activity is still present.
[0126] In addition to the detecting Bcr-Abl, the present invention
provides a fast, quantitative, and reliable detection method for
other oncogenic tyrosine kinases. This should greatly improve
patient diagnosis and care. For example, CML is a cancer that is
easily detectable; the 9:22 chromosome translocation can be seen
using cytogenetics. However, most other forms of cancers are not so
easily diagnosed. For these cancers, the underlying causes are not
as easily identified. The present invention can be used to identify
Her-2/neu activity in breast cancer, FGFR activity in lung and
ovary cancer, Raf-kinase activity in bladder and colon cancer,
c-Met activity in renal cancer, and c-Kit activity in gastric
cancer [28, 29, 31].
[0127] While most of the above Examples were performed using
protein-acrylamide copolymer spots approximately 2 mm in diameter
(1 .mu.l spots), Example 10 demonstrates the ability to create
surface spots several orders of magnitude smaller. FIG. 11A
provides fluorescence images of spots printed from a micropipette
with GST-GFP concentrations of 0, 95, and 470 .mu.g/ml and
spot-to-spot spacing of 500, 750, and 1000 .mu.m. From this image
it can be seen that the acrylamide-based surface attachment is
reproducible and the surface-attached spots are approximately 300
to 400 .mu.m in size. Distribution of the GFP fluorescence signal
throughout the volume of the spots can be seen in FIG. 11B.
[0128] A fluorescence image of surface-attached GST-GFP spots
printed with a spot-to-spot spacing of 250 .mu.m is shown in FIG.
12. Each spot (or domain) is approximately 150 .mu.m in size.
Currently, reduction of the domain size below 150 .mu.m in diameter
is limited solely by the micropipettes used for the printing and
not the polyacrylamide-based surface attachment strategy. Thus,
domains considerably smaller than 150 .mu.m can be fabricated.
Example 11
Array-Based Detection of Abl Tyrosine Kinase Activity from Cell
Lysates
[0129] Preparation of Abl Substrates: GST-CRKL fusion constructs
were prepared as 5 described in Example 2, supra. GST-CRKL (full
length), GST-CRKL (SH3), and GST-CRKL (intra SH3) were transformed
into Escherichia coli BL21 cells and grown to mid-log phase at
37.degree. C. in 2.times.YT (16 g tryptone, 10 g yeast extract, 5 g
NaCl, pH 6.6 in 1 l of water). Protein expression was induced by
addition of isopropyl-.beta.-D-thiogalactopyranoside (IPTG) to a
final concentration of 0.1 mM. After three hours, cells were
harvested, resuspended in lysis buffer (140 mM NaCl, 2.7 mM KCl, 10
mM Na.sub.2HPO.sub.4, 1.7 mM KH.sub.2PO.sub.4, 0.5 mM DTT, 1 mM
sodium orthovandate, 1 mM PMSF, 4% 25.times.complete protease
inhibitor (Roche Diagnostics GmbH, Penzberg, Germany), 1% Triton
X-100, pH 7.4), and lysed on ice. Cell lysate was purified by
affinity chromatography following the manufacture's instructions on
a glutathione sepharose column (Amersham Biosciences, Piscataway,
N.J.). Following addition of cell lysate to the column, the column
was washed with PBS, pH 7.3 until the flow through was
protein-free. The GST-Crkl fusion protein was then eluted by
addition of 50 mM Tris-HCl, pH 8.0 containing 10 mM reduced
glutathione. Purified protein was concentrated in a centrifugal
filter (Millipore, Billerica, Mass.) with a 10 kDa nominal
molecular weight cutoff. TABLE-US-00001 TABLE 1 Description of
Fusion Proteins Notation Crkl amino acids Crkl functional domains
GST-Crkl (full length) 1-303 SH2-SH3-*-SH3 GST-Crkl (SH3) 120-303
SH3-*-SH3 GST-Crkl (intra SH3) 180-245 -*- *represents tyrosine
phosphorylation site for Abl/Bcr-Abl.
[0130] Preparation of K562 and HL60 Cell Lysates: K562 and HL60
cells (ATCC, Manassas, Va.) were cultured at 37.degree. C. and 5%
CO.sub.2 in RPMI-1640 media (Cambrex Bio Science, Walkersville, MD)
containing 100 units/ml penicillin, 100 .mu.g/ml streptomycin, and
10% FBS. For lysis, cells were resuspended at 5.times.10.sup.7
cells/ml in lysis buffer (42.3 mM HEPES, 126 mM NaCl, 1.27 mM
MgCl.sub.2, 0.85 mM EDTA, 84.5 mM NaF, 8.45 mM sodium
pyrophosphate, 0.169 mM sodium orthovanadate, 1 mM PMSF, 0.95%
Triton X-100, 9.5% glycerol, 4% 25.times.complete protease
inhibitor, pH 7.4) and incubated on ice for 20 min. The total cell
lysate was then clarified by spinning at 1500 rpm for 10 min. Total
protein concentration was determined via a Pierce BCA protein assay
kit (Pierce, Rockford, Ill.) and cell lysates were stored at
-80.degree. C. until further use.
[0131] Bead-Based Kinase Assays. SwellGel Discs (Pierce) were
suspended in cold 50 mM Tris, pH 7.5 so that 1 .mu.l of bead
suspension bound 1 .mu.g of GST fusion protein. One nmol of
GST-Crkl substrate was incubated with the glutathione bead
suspension for 1 h at 4.degree. C. with constant rotation. The
substrate-bound beads were washed twice with ice-cold 50 mM
Tris-HCl, pH 7.5 containing 10 mM MgCl.sub.2. Substrate-bound beads
were then incubated with either recombinant v-Abl or K562 cell
lysate. The v-Abl reaction mixtures contained: 20 .mu.l
4.times.buffer (200 mM Tris-HCl, 40 mM MgCl.sub.2, 4 mM DTT, pH
7.5); 20 .mu.l 40 .mu.M ATP; 0.5 .mu.l v-Abl (EMD Biosciences,
Inc., San Diego, Calif.); 0 or 20 .mu.l 400 .mu.M imatinib; and
water to a total volume of 80 .mu.l. The K562 cell lysate reaction
mixtures contained: 20 .mu.l 4.times.buffer; 20 .mu.l 40 .mu.M ATP;
50 .mu.g K562 cell lysate; 0 or 20 .mu.l 400 .mu.M imatinib; and
water to a total volume of 80 .mu.l. The reactions allowed to
proceed for 1 h at 30.degree. C. Following the reaction, the beads
were washed twice with ice-cold 50 mM Tris, pH7.5. GST-Crkl
substrates were eluted with 10 mM reduced glutathione in 50 mM
Tris-HCl, pH 8.0. Kinase assay samples were loaded in a 12%
SDS-PAGE gel and transferred to nitrocellulose membranes according
to standard procedures. Consistent sample loading was verified
using the Memcode Reversible Protein Stain Kit (Pierce). Membranes
were probed with anti-phosphotyrosine antibodies.
[0132] Fabrication of Protein Arrays: Glass slides were
acrylic-functionalized and GST-Crkl fusion proteins were
acrylic-labeled as described in the previous Examples. Briefly,
fusion proteins were labeled on primary amines by reaction with
6-((acrylo)amino) hexanoic acid, succinimidyl ester (Molecular
Probes, Eugene, Oreg.) and clean glass slides were functionalized
via reaction with (3-acryloxypropyl)-trimethoxysilane (Gelest,
Tullytown, PA).
[0133] Following functionalization of the glass slides, a thin
layer of 18.8% polyacrylamide gel was attached to the glass
surface. Forty .mu.l of a mixture containing 125 .mu.l 1.5 M Tris,
pH 8.8; 285 .mu.l 33% acrylamide mix (0.86 g
N,N'-methylenebisacrylamide (Bis) and 32.14 g acrylamide in a total
volume of 100 ml); 5 .mu.l 10% ammonium persulfate (APS); 75 .mu.l
100% glycerol; 0.5 .mu.l N,N,N',N'-tetramethylethylenediamine
(TEMED); and 9.5 .mu.l water was sandwiched between an
acrylic-functionalized glass slide and a clean glass plate and
allowed to polymerize in a N.sub.2 environment at room temperature
for 30 min. The glass plate was then removed and 1 .mu.l protein
spots were polymerized on top of the 18.8% acrylamide base layer
from the following mixture: 6.25 .mu.l 1.5 M Tris, pH 8.8; 3 .mu.l
33% acrylamide mix; 0.5 .mu.l 10% APS; 3.75 .mu.l 100% glycerol;
0.1 .mu.l TEMED; 0 to 7.5 .mu.l acrylic-labeled GST-Crkl protein
solution; and water to a total volume of 25 .mu.l. Additionally, 3
.mu.l spacer spots of the above mixture without protein were added
on the exterior corners of the protein array for the subsequent
creation of a reaction chamber as described below. After attachment
of the protein-acrylamide copolymer spots the slides were washed by
briefly dipping into approximately 250 ml of TBST (10 mM Tris-HCl,
100 mM NaCl, 0.1% Tween-20, pH 7.5) followed by a 15 minute and two
5 minute washes with slight agitation in approximately 20 ml of
TBST. Slides were then washed by briefly dipping into approximately
250 ml of water followed by two 5 minute washes with slight
agitation in approximately 20 ml of water, before being stored
overnight at 4.degree. C. in Abl kinase assay buffer (50 mM
Tris-HCl, 10 mM MgCl.sub.2, 100 .mu.M EDTA, 1 mM DTT, 0.015% Brij
35, 100 .mu.g/ml BSA, pH 7.5).
[0134] Array-Based Kinase Assays: Just prior to the kinase assay,
the glass slides were removed from the Abl kinase assay buffer and
dried under a stream of compressed air. Careful attention was paid
to make sure that only the bare portions of the slide were dried
and the polyacrylamide spots and polyacrylamide base layer remained
hydrated. Using the 3 .mu.l spacer spots, a reaction chamber was
then created by suspending a clean glass slide over top of the 1
.mu.l protein-acrylamide copolymer hydrogel spots. The v-Abl
reaction mixtures contained: 100 .mu.l 3.times.Abl kinase assay
buffer (150 mM Tris-HCl, 30 mM MgCl.sub.2, 300 .mu.M EDTA, 3 mM
DTT, 0.045% Brij 35, 300 .mu.g/ml BSA, pH 7.5); 30 .mu.l 1 mM ATP;
50 .mu.l 100% glycerol; 1.5 .mu.l v-Abl; 0 to 100 .mu.l 30 .mu.M
imatinib; and water to a total volume of 300 .mu.l. The K562 cell
lysate reaction mixtures contained: 100 .mu.l.times.Abl kinase
assay buffer; 3 .mu.l 1 mM ATP; 50 .mu.l 100% glycerol; 0 to 450
.mu.g K562 cell lysate; 0 to 450 .mu.g HL-60 cell lysate; 0 to 30
.mu.l 3 mM imatinib; and water to a total volume of 300 .mu.l.
Aliquots (250 .mu.l) of these kinase reaction mixtures were then
applied to each reaction chamber and the reactions allowed to
proceed for 30 min to 5 h at 30.degree. C. in a saturated
environment. After the reaction, the glass slide used to create the
reaction chamber was removed and the protein array was washed by
briefly dipping into approximately 250 ml TBST followed by a 15
minute and two 5 minute washes with slight agitation in
approximately 20 ml of TBST.
[0135] Tyrosine kinase inhibitors AG1478, PP2, AG1296, and AG490
(Calbiochem, San Diego, Calif.) and PKI166 (Novartis, Basel,
Switzerland) were dissolved in DMSO at 50.times.final
concentration. Experiments with these inhibitors were conducted as
above and contained: 50 .mu.l 3.times.Abl kinase assay buffer; 1.5
.mu.l 1 mM ATP; 25 .mu.l 100% glycerol; 225 .mu.g K562 cell lysate;
3 .mu.l 50.times.inhibitor in DMSO; and water to a total volume of
150 .mu.l. Aliquots (100 .mu.l) of this reaction mixture were
applied to each reaction chamber (this time created with a clean
glass cover slip rather than a glass slide) and the reaction
allowed to proceed for 2 h at 30.degree. C. in a saturated
environment.
[0136] Chemiluminescence Detection of Phosphorylated Substrates:
Slides were blocked in approximately 20 ml of TBST containing 1%
BSA for 1 hour at room conditions. Each slide was then removed from
the blocking solution and incubated with 1.5 .mu.g of monoclonal
anti-phosphotyrosine antibody PY20 (Sigma, Saint Louis, Mo.) in 1.5
ml of TBST for 1 hour. Each slide was then washed by briefly
dipping into approximately 250 ml TBST followed by a 15 minute and
two 5 minute washes with slight agitation in approximately 20 ml of
TBST. Each slide was then incubated with 0.15 .mu.g of horseradish
peroxidase (HRP)-conjugated secondary antibody (Molecular Probes,
Eugene, Oreg.) in 1.5 ml of TBST for 1 hour and subsequently washed
in TBST as above. The slides were then stored in TBST until
detection.
[0137] Enhanced chemiluminescence (ECL) Western blotting detection
reagent (Amersham Biosciences) was used to detect horseradish
peroxidase-labeled slides. Briefly, after removal from the wash
buffer each slide was treated with 1.5 ml of ECL detection reagent
for 1 min. The detection reagent was then "shaken off" from the
slide and the slide was placed between transparent acetate sheets
for subsequent exposure to film (Amersham Biosciences). After
development, the average gray value of each spot was obtained using
ImageJ software (NIH, Bethesda, Md.).
Results and Discussion of Example 11:
[0138] Abl Kinase Activity Toward Bead-Immobilized Substrates: To
identify a suitable substrate for assessing Abl kinase activity on
a surface, a Western blot analysis was used to measure
phosphorylation of several GST-Crkl constructs immobilized to
glutathione beads. Three GST-Crkl fusion proteins were used in this
work, GST-Crkl (full length), GST-Crkl (SH3), and GST-Crkl (intra
SH3). These proteins contain GST fused to full length Crkl, a Crkl
fragment containing both SH3 domains, and a Crkl fragment
containing only the sequence immediately surrounding the Y207 Abl
phosphorylation site. Phosphorylation of GST-Crkl substrates by
purified v-Abl and in Bcr-Abl-containing K562 cell extracts, in the
presence and absence of the Abl kinase inhibitor imatinib mesylate,
is shown in FIGS. 13A and 13B, respectively. v-Abl displays
tyrosine kinase activity toward GST-Crkl (SH3) and GST-Crkl (intra
SH3) in the absence of imatinib, and kinase activity towards these
substrates is minimal at 100 PM imatinib. GST-Crkl (full-length) is
not phosphorylated by v-Abl. In the K562 cell lysate system, all
GST-Crkl substrates are phosphorylated in the absence of imatinib.
However, only the phosphorylation of the GST-Crkl (full-length) and
GST-Crkl (SH3) substrates is inhibited at 100 .mu.M imatinib. As
imatinib is a specific Abl inhibitor, the lack of inhibition of
GST-Crkl (intra SH3) phosphorylation in the cell lysate system is
likely due to phosphorylation by other tyrosine kinases contained
within the cell lysate. Presumably, portions of the SH3 domain
prevent tyrosine phosphorylation by these nonspecific kinases. Due
to this decrease in specificity towards Abl kinase, the GST-Crkl
(intra SH3) substrate was not used in subsequent protein array
studies of the K562 cell lysate.
[0139] Fabrication of GST-Crkl Arrays: Protein arrays for the
surface-based detection of Abl kinase activity were created using
the protein-acrylamide copolymerization attachment scheme described
in the previous Examples. Briefly summarized, clean glass slides
were treated with (3-acryloxpropyl)-trimethoxysilane, forming an
acrylic-functionalized surface. Free acrylamide monomer and the
crosslinker Bis were then polymerized to the acrylic-functionalized
surface, resulting in a polyacrylamide- coated surface. This
approximately 20 .mu.m thick polyacrylamide layer greatly reduces
the background signal resulting from nonspecific binding of cell
extract components to bare glass during the Bcr-Abl kinase
reactions (data not shown). GST-Crkl fusion proteins were also
acrylic-functionalized via reaction with 6-((acrylo)amino) hexanoic
acid, succinimidyl ester. Acrylic-functionalized GST-Crkl fusion
proteins were then mixed with free acrylamide monomer and
crosslinker and spotted onto the polyacrylamide coated surface.
Polymerization results in the incorporation of GST-Crkl into
polyacrylamide gel spots, which spots are in turn linked to the
polyacrylamide coated glass slide.
[0140] Protein Array-Based Detection of Purified v-Abl Activity:
Initial surface-based studies investigated the activity and
inhibition of Abl in a purified system. GST-Crkl substrate arrays
were incubated in the presence of 100 .mu.m ATP and purified v-Abl
and phosphorylated Crkl was detected via anti-phosphotyrosine
antibodies (see FIGS. 14A and 14B, respectively). In a 2 h
reaction, v-Abl demonstrates a slight preference for the GST-Crkl
(SH3) substrate relative to the GST-Crkl (intra SH3) substrate
(FIG. 14A). Phosphorylation of GST-Crkl (full-length) is minimal
(data not shown). As expected, time course data for the
phosphorylation of GST-Crkl (SH3) show an increase in Crkl
phosphorylation with increasing reaction time (FIG. 14B).
[0141] Data for the inhibition of v-Abl by imatinib can be seen in
FIG. 15. Half-maximal inhibition (IC.sub.50) values for the
inhibition of GST-Crkl (intra SH3) and GST-Crkl (SH3)
phosphorylation by imatinib are 2.0 and 1.5 .mu.M respectively.
[0142] Protein Array-Based Detection of Bcr-Abl Activity in a Cell
Lysate: After demonstrating the ability to measure v-Abl activity
and inhibition in a purified system, the Bcr-Abl activity in cell
lysates was measured. Direct kinase assays from cell lysates are
very desirable from a time and cost perspective in diagnostics
development; however, the complex composition of cell lysates often
complicates data analysis and leads to false positives or
negatives. Extracts from CML cells contain numerous phosphorylated
proteins, including endogenous phosphorylated Crkl. Thus,
minimization of the nonspecific surface binding and subsequent
detection of these phosphorylated components is critical. In
addition to Bcr-Abl, the cell lysate is also expected to contain
multiple tyrosine kinases. Phosphorylation of surface-immobilized
Crkl by these additional kinases could obscure the specific
detection and quantification of Bcr-Abl activity. Not only is a
cell lysate likely to contain additional kinases, but it is also
expected to contain phosphatases. Phosphatase-catalyzed
dephosphorylation of surface-immobilized phosphorylated-Crkl would
result in a lower estimation of Bcr-Abl activity than is actually
present. Elimination of nonspecific binding was accomplished
through the use of a polyacrylamide coated glass surface as
disclosed herein.
[0143] In initial K562 cell lysate studies, GST-Crkl substrate
arrays were incubated with 10 .mu.M ATP and 375 .mu.g of K562 cell
lysate. Thirty minute reaction data show a strong preference for
phosphorylation of GST-Crkl (SH3) relative to GST-Crkl (full
length). See FIGS. 16A and 16B. Time course data for the
phosphorylation of GST-Crkl (SH3) display the expected increase in
phosphorylation with an increase in reaction time; however, in
contrast to v-Abl phosphorylation the reaction appears to be nearly
complete after 30 min (FIG. 16B). This is likely due to a high
level of Bcr-Abl activity in the cell extracts. Imatinib inhibition
of both GST-Crkl (SH3) and GST-Crkl (full length) phosphorylation
by K562 cell lysates occurs with an IC.sub.50 value of
approximately 30 PM (see FIG. 17). Because imatinib is an ATP
competitive inhibitor, this increase in the IC.sub.50 value in
respect to the v-Abl system may be due to the cell lysate
containing a physiological level of ATP. Also, as imatinib is a
relatively specific inhibitor of Bcr-Abl, this data supports the
notion that the measured tyrosine kinase activity of the cell
lysate toward the immobilized Crkl constructs is indeed due to
Bcr-Abl.
[0144] Given the complexity of the K562 cell extract, experiments
were conducted to assess the potential influence of phosphatases
within the cell lysate on the resulting level of GST-Crkl
phosphorylation. Protein arrays containing 165 ng/mm.sup.2 spots of
GST-Crkl (SH3) were phosphorylated for 2 h in the presence of 10
.mu.M ATP and 375 .mu.g of K562 cell lysate. Following thorough
washing, the surfaces were incubated in the reaction buffer without
K562 extract, with K562 extract, and with K562 extract and 300
.mu.M imatinib. These reactions represent a negative control, the
potential for phosphatase and Abl kinase activity in the cell
extract, and the potential for only phosphatase activity in the
cell extract, respectively. Data indicate that the level of Crkl
phosphorylation between these three samples did not significantly
change with reaction times up to 2 h (see FIG. 18). Also, only a
slight decrease in Crkl phosphorylation is observed after 5 h,
suggesting minimal phosphatase activity in the K562 cell lysates
toward the phosphorylated, immobilized GST-Crkl substrate.
[0145] To assess the lower detection limit of the
protein-acrylamide copolymer arrays, Bcr-Abl activity was
quantified in samples of K562 cell lysate (Bcr-Abl.sup.+) diluted
into HL60 cell lysate (Bcr-Abl.sup.-). Protein arrays containing
495 ng/mm.sup.2 spots of GST-Crkl (SH3) were incubated for 2 h in
reaction mixtures containing 375 total .mu.l cell lysate, 0 to 100%
of that derived from K562 cells and the remainder from HL60 cells.
FIG. 19 demonstrates that Bcr-Abl activity is detectable at 2.5%
K562 extract, with statistically significant results obtains for
levels at and above 15% K562 extract (P value of 0.02).
Additionally, the level of Crkl phosphorylation in the pure HL60
cell lysate is only 3.5% of that observed in the pure K562 cell
lysate. Because both K562 and HL60 cell lysates are expected to
contain a variety of active tyrosine kinases, but only the K562
cell lysate is expected to contain Bcr-Abl, these data again
support the notion that the tyrosine kinase activity toward the
immobilized GST-Crkl substrates is indeed due to Bcr-Abl.
[0146] Screening Chemical Inhibitors of Bcr-Abl Activity Toward
Arrayed Crkl Substrates. While the Examples discussed above
demonstrate the ability of the protein-acrylamide copolymer
hydrogel arrays to detect v-Abl and Bcr-Abl activity reproducibly
and quantitatively, a significant advantage of array technology can
be found in the high-throughput nature in which multiple data
points can be simultaneously obtained. Thus, in addition to
quantifying Bcr-Abl activity from cell extracts as a diagnostic
tool, arrays of immobilized Crkl substrates can also used as a
platform for identifying and characterizing novel inhibitors of
kinase activity. In order to demonstrate this potential, Bcr-Abl
activity was measured in the presence of varying concentrations of
six different tyrosine kinase inhibitors (see Table 2). Protein
arrays containing 165 ng/mm.sup.2 spots of GST-Crkl (SH3) were
incubated for 2 h in reaction mixtures containing K562 cell lysate
and tyrosine kinase inhibitor concentrations ranging from 100 nM to
1 mM. Significant inhibition of Bcr-Abl activity can be seen with
PKI166, AG1478, and PP2 at 100 .mu.M and 1 mM concentrations (see
FIG. 20). Thus, the resulting IC.sub.50 values for these inhibitors
are in the 100 .mu.M range. AG1296 and AG490 do not inhibit Bcr-Abl
activity over the range studied (IC.sub.50>1 mM). TABLE-US-00002
TABLE 2 Inhibitor Target Mode of action imatinib Abl, c-Kit, PDGFR
ATP competitive PKI166 EGFR ATP competitive AG1478 EGFR ATP
competitive PP2 p56.sup.lck, p59.sup.fynT ATP competitive AG1296
PDGFR ATP competitive AG490 JAK-2 ATP competitive
[0147] In addition to eliminating the electrophoresis and transfer
steps of the Western blot, the array format of disclosed herein
allows simultaneous detection of multiple signals. In the present
Examples, up to 40 GST-Crkl spots per microscope slide have been
detected; however, with commercially-available microarray printing
techniques densities as high as thousands of spots per slide are
possible.
[0148] This Example is significant because it demonstrates that the
present invention can directly measure the tyrosine kinase activity
of an oncogenic moiety. Therefore, the present invention can
directly measure imatinib resistance due to BCR-ABL transcript
overexpression, and also directly measure imatinib resistance due
to mutation of the Bcr-Abl protein. Additionally, the ability to
detect protein activity and inhibition extends beyond the imatinib
system. By simply replacing imatinib with other inhibitors, data
for multiple tyrosine kinase inhibitors at multiple concentrations
can be simultaneously obtained. Thus, the protein-acrylamide
copolymerization strategy disclosed hereing provides a platform on
which high-throughput screening assays can be implemented for other
biological systems.
[0149] In contrast to many other protein array systems, the present
invention takes advantage of a hydrogel-based covalent surface
immobilization. Polyacrylamide and protein-acrylamide copolymer
hydrogels exhibit low levels of nonspecific protein adsorption.
Thus, the subsequent detection of nonspecifically bound proteins
from cell extracts is limited. This reduction in nonspecific
binding has been shown to produce a six-fold increase in the
signal-to-noise ratio obtained from polyacrylamide protein arrays
vs. poly-L-lysine protein arrays in a human serum diagnostic. In
the present invention, the inclusion of a polyacrylamide layer
between the glass slide and protein array spots greatly reduced the
nonspecific binding of cell lysate components and minimized the
detection of endogenbus phosphorylated-Crkl or other
phosphoproteins. In addition to minimizing nonspecific protein
adsorption, the protein-acrylamide copolymerization strategy
maintains the immobilized proteins within a hydrophilic
environment. This environment prevents protein dehydration and
minimizes denaturation due to hydrophobic and/or charged
protein-surface interactions. Also, the covalent nature of the
protein-acrylamide copolymerization strategy ensures stable
immobilization of proteins, even over extended periods of protein
array storage.
[0150] The porous, three-dimensional shape of the
protein-acrylamide copolymer array spots provides several
advantages over traditional, two-dimensional protein immobilization
techniques. The capacity of the hemispherical spot is greatly
increased as compared to strategies in which proteins are directly
attached to the solid surface. The three-dimensional structure of
the spot also positions a majority of the immobilized proteins away
from the surface. Thus, immobilized proteins are more likely to be
assessable to the sample applied to the protein array.
Additionally, well-established chemistry exists for controlling the
porosity and pore size distribution within polyacrylamide gels. By
simply changing the polymerization conditions, a protein-acrylamide
copolymer hydrogel array in which each protein is immobilized
within a different porosity gel spot can be created. In the present
Example, the polymerization conditions were purposefully chosen to
provide the least possible cross-linking while still maintaining a
mechanically stable hydrogel. These conditions provided maximum
accessibility of the relatively large Bcr-Abl kinase (190 kD) to
the immobilized substrate (data not shown).
[0151] While the Examples disclosed herein used antibody-based
chemiluminescence detection of surface-attached substrates,
additional detection techniques can be used in the present
invention. For example, by replacing HRP-conjugated antibodies with
fluorescently labeled antibodies, multiple protein-state specific
signals can be simultaneously detected on the protein-acrylamide
copolymer array. Non antibody-based detection techniques are also
within the scope of the present invention, including detection of
surface-attached substrates via a small molecule, phospho-specific
fluorescent dye has been demonstrated, gel tryptic digestion
followed by mass spectrometry, or mass spectrometry directly from
polyacrylamide gels.
REFERENCES
[0152] [1] Li, Y., Tang, Y., Ye, L., Liu, B., Liu, K., Chen, J.,
and Xue, Q. (2003) J Cancer Res Clin Oncol 129:43-51. [0153] [2]
Onody, A., Zvara, A., Hackler, L., Jr., Vigh, L., Ferdinandy, P.,
and Puskas, L. G. (2003) FEBS Lett 536:35-40. [0154] [3] Dubnau,
J., Chiang, A. S., Grady, L., Barditch, J., Gossweiler, S., McNeil,
J., Smith, P., Buldoc, F., Scott, R., Certa, U., Broger, C., and
Tully, T. (2003) Curr Biol 13:286-96. [0155] [4] Gygi, S. P.,
Rochon, Y., Franza, B. R., and Aebersold, R. (1999) Mol Cell Biol
19:1720-30. [0156] [5] Espejo, A., Cote, J., Bednarek, A., Richard,
S., and Bedford, M. T. (2002) Biochem J 367:697-702. [0157] [6]
Houseman, B. T., Huh, J. H., Kron, S. J., and Mrksich, M. (2002)
Nat Biotechnol 20:270-4. [0158] [7] Haab, B. B., Dunham, M. J., and
Brown, P. O. (2001) Genome Biol 2. [0159] [8] MacBeath, G., and
Schreiber, S. L. (2000) Science 289:1760-3. [0160] [9] Salisbury,
C. M., Maly, D. J., and Eliman, J. A. (2002) J Am Chem Soc
124:14868-70. [0161] [10] Seong, S. Y. (2002) Clin Diagn Lab
Immunol 9:927-30. [0162] [11] Fang, Y., Frutos, A. G., and Lahiri,
J. (2002) Chembiochem 3:987-91. [0163] [12] Zhu, H., Bilgin, M.,
Bangham, R., Hall, D., Casamayor, A., Bertone, P., Lan, N., Jansen,
R., Bidlingmaier, S., Houfek, T., Mitchell, T., Miller, P., Dean,
R. A., Gerstein, M., and Snyder, M. (2001) Science 293:2101-5.
[0164] [13] Zhu, Q., Uttamchandani, M., Li, D. B., Lesaicherre, M.
L., and Yao, S. Q. (2003) Organic Letters 5:1257-1260. [0165] [14]
Fahy, E., Davis, G. R., DiMichele, L. J., and Ghosh, S. S. (1993)
Nucleic Acids Res 21:1819-26. [0166] [15] Guschin, D., Yershov, G.,
Zaslavsky, A., Gemmeil, A., Shick, V., Proudnikov, D., Arenkov, P.,
and Mirzabekov, A. (1997)Anal Biochem 250:203-11. [0167] [16]
Arenkov, P., Kukhtin, A., Gemmell, A., Voloshchuk, S., Chupeeva,
V., and Mirzabekov, A. (2000) Anal Biochem 278:123-31. [0168] [17]
Rubina, A. Y., Dementieva, E. I., Stomakhin, A. A., Daril, E. L.,
Pankov, S. V., Barsky, V. E., Ivanov, S. M., Konovalova, E. V., and
Mirzabekov, A. D. (2003) Biotechniques 34:1008-14, 1016-20, 1022.
[0169] [18] Songyang, Z., Carraway, K. L., 3rd, Eck, M. J.,
Harrison, S. C., Feldman, R. A., Mohammadi, M., Schiessinger, 1,
Hubbard, S. R., Smith, D. P., Eng, C., et al. (1995) Nature
373:536-9. [0170] [19] Vasiliskov, A. V., Tiniofeev, E. N.,
Surzhikov, S. A., Drobyshev, A. L., Shick, V. V., and Mirzabekov,
A. D. (1999) Biotechniques 27:592-4, 596-8, 600 passim. [0171] [20]
Berman, H. M., Westbrook, J., Feng, Z., Gilliland, G., Bhat, T. N.,
Weissig, H., Shindyalov, I. N., and Boume, P. E. (2000) Nucleic
Acids Res 28:235-42. [0172] [21] Houseman, B. T., and Mrksich, M.
(2002) Trends Biotechnol 20:279-81. [0173] [22] Falsey, J. R.,
Rend, M., Park S., Li, S., and Lam, K. S. (2001) Bioconjug Chem
12:346-53. [0174] [23] Nicholson, J. K., and Wilson, I. D. (2003)
Nature Reviews Drug Discovery 2: 668-676. [0175] [24] Shipkova, M.,
Armstrong, V. W., Oellerich, M., and Wieland, E. (2003) Therapeutic
Drug Monitoring 25: 1-16. [0176] [25] Righetti, P. G., and Gelfi,
C. (1997) J of Chromatography B 699:63-75. [0177] [26] Patras, G.,
Qiao, G. G., and Solomon, D. H. (2001) Electrophoresis
22:4303-4310. [0178] [27] Deininger, M. W. N., and Druker, B. J.
(2003) Pharmacological Reviews 55:401-423. [0179] [28] Sawyers, C.
L. (2002) Current Opinion in Genetics & Development 12;1
11-115. [0180] [29] Hubbard, S. R. (2002) Current Opinion in
Structural Biology 12:735-741. [0181] [30] Griffin, J. (2001)
Seminars in Oncology 28:3-8. [0182] [31] Traxler, P., Bold, G.,
Buchdunger, E., Caravatti, G., Furet, P., Manley, P., O'Reilly, T.,
Wood, J., and Zimmermann, J. (2001) Medicinal Research Reviews
21:499-512. [0183] [32] George, D. (2001) Seminars in Oncology
28:27-33. [0184] [33] Demetri, G. D. (2001) Seminars in Oncology
28:19-26. [0185] [34] Clarkson, B., Strife, A., Wisniewski, D.,
Lambek, C. L., and Liu, C. (2003) Leukemia 17:1211-1262. [0186]
[35] Nowell, P., and Hungerford, D. (1960) J Natl Cancer Inst
25:85-109. [0187] [36] Druker, B. J., Talpaz, M., Resta, D. J.,
Peng, B., Buchdunger, E., Ford, J. M., Lydon, N. B., Kantaijian,
H., Capdeville, R., Ohno-Jones, S., and Sawyers, C. L. (2001) New
England Journal of Medicine 344:1031-1037. [0188] [37] Hehlmann, R.
(2003) Leukemia 17:1010-1012. [0189] [38] von Bubnoff, N., Peschel,
C., and Duyster, J. (2003) Leukemia 17:829-838. [0190] [39] Heaney,
C., Kolibaba, K., Bhat, A., Oda, T., Ohno, S., Fanning, S., and
Druker, B. J. (1997) Blood 89:297-306. [0191] [40] Senechal, K.,
Halpern, J., and Sawyers C. L. (1996) J Biol Chem 271(38):23255-61.
Sequence CWU 1
1
3 1 9 PRT Artificial Artificial Sequence - C-terminus of
glutathione-S-transferase fusion peptide 1 Glu Glu Glu Ile Tyr Gly
Glu Phe Glu 1 5 2 53 DNA Artificial Artificial consensus sequence
for c-Src tyrosine phosphorylation 2 aaaaaaagga tccgaagaag
aaatttatgg ggaattcgaa gaattccccc ccc 53 3 15 PRT Artificial Amino
acid sequence of isolated c-Src consensus sequence 3 Ala Glu Glu
Glu Ile Tyr Gly Glu Phe Glu Ala Lys Lys Lys Lys 1 5 10 15
* * * * *